Myceliated  cacao products and methods for making myceliated products from green cacao and other agricultural substrates

ABSTRACT

The present invention provides a method for the preparation of a myceliated cacao bean or other agricultural product. This method includes providing cacao beans or other agricultural substrate, optionally hydrating the cacao beans or other agricultural product, and optionally pasteurizing or sterilizing the cacao beans or other agricultural substrate to provide prepared cacao beans or other agricultural substrate, and a step of inoculating the prepared cacao beans or other agricultural substrate with a prepared fungal component and culturing the inoculated cacao beans or other agricultural substrate to prepare the myceliated product. The methods of the instant invention result in prepared cacao beans or other agricultural substrate having reduced levels of undesirable taste components, such as theobromine, catechin, epicatechin, gallic acid equivalents, and/or 2-methoxy-3-isopropylpyrazine, and increased levels of myceliation products, such as fungal β-glucans, chitin, proteins, glycoproteins, pyrazines and polysaccharides, relative to starting cacao beans or other agricultural substrate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/953,823 entitled “Myceliated Products and Methods for MakingMyceliated Products from Cacao and Other Agricultural Substrates”, filedMar. 15, 2014; this application also claims priority as a continuationto pending WIPO application PCT/US14/29998, filed Mar. 15, 2014,entitled “Myceliated Products And Methods For Making Myceliated ProductsFrom Cacao And Other Agricultural Substrates,” and the disclosure ofeach is hereby incorporated by reference herein in their entirety.

TECHNICAL FIELD

The methods and products relate to use of fungal strains andmycotechnological methods to improve flavor in agricultural substrates,in particular, cacao and other agricultural substrates.

BACKGROUND

Most of the world's cacao is grown in a band spanning 20 degrees oflatitude north and south of the equator, as the cacao tree (Theobromacacao) needs a humid, hot tropical environment in which to grow andfruit healthy cacao pods that contain the cacao bean. Harvested whenripe, the average cacao pod produces 20 to 50 cacao beans emulsed inmucilaginous pulp. Generally, cacao beans are prepared for the market byfermenting the beans for four to seven days (to remove the pulp, a stepalso known as demucilaging which may be accomplished by other methodsknown in the art) and the beans are dried for another five to fourteendays. There are about 20 different species of cacao and hundreds ofhybrids. Of these, four major varietals dominate the commercialchocolate market. Forastero cacao accounts for approximately 90% of theworld's cacao market. Criollo cacao accounts for about 7% of the globalmarket, and is characterized as a finer, gourmet cacao that is moredifficult to grow than Forastero. Trinitario cacao is aForastero/Criollo hybrid that grows well in Trinidad. Nacional cacao isprized for having low bitterness and a sweet floral aroma, but isdifficult to grow commercially. A majority of the world's cacao isproduced in the Ivory Coast. This volume represents over 1 million tonsper year. Ghana, Indonesia, Cameroon, Brazil, and Nigeria produce majoramounts of cacao as well. Cacao is consumed world-wide, usually in theform of chocolate, which is a mixture solidified cacao liquor andsweeteners, among other components.

It is common for cacao beans to be treated with superheated steam tomitigate the bacterial content. It is believed that treating cacao beanswith saturated steam also removes some of the desirable flavorcomponents. Sugars and other sweeteners are mixed with cacao powder toform good-tasting chocolate. Often other flavors are added. Currentlymany health advocates, while acknowledging the health benefits of cacao,warn against the risks of excessive use of sweeteners including sugarand artificial sweeteners.

What is desired is a way of manufacturing cacao that achieves a greattasting product without the need for excessive sweetening. What is alsodesired is a way of processing cacao that retains desirable flavorcomponents while minimizing the less desirable flavor components.Further, it is desirable to rely on natural processes to achieve a greattasting product. Cacao is a complex food reported to contain over 20,000different chemical compounds, making this a complex and hard-to-predicttask.

A need remains in the art for improved cacao products having reducedlevels of undesirable taste components and/or increased levels of flavorand/or health promoting components relative to cacao beans or otheragricultural substrate, and for methods of obtaining such products.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a method for thepreparation of a myceliated cacao or other agricultural substrateproduct. This method includes the step of providing prepared cacao beansor other agricultural substrate, which includes providing cacao beans orother agricultural substrate, optionally hydrating the provided cacaobeans or other agricultural substrate, and sterilizing and/orpasteurizing the cacao beans or other agricultural substrate to provideprepared cacao beans or other agricultural substrate. The method alsoincludes the step of providing a prepared fungal component. The methodalso comprises inoculating the prepared cacao beans or otheragricultural substrate with the prepared fungal component and culturingthe inoculated cacao beans or other agricultural substrate to preparethe myceliated cacao or other agricultural substrate product.

In one embodiment, the method includes reducing the amount ofundesirable taste components. Reducing the amount of undesirable tastecomponents optionally includes at least one, or two aqueous extractionsof the cacao beans or other agricultural substrate. An undesirable tastecomponent includes catechins, theobromine and/or2-methoxy-3-isopropylpyrazine.

In one embodiment, the method includes a step of hydrating the cacaobeans or other agricultural substrate, to a moisture content of 20 to80%, optionally to about 40%. The methods of the instant invention alsoinclude where the prepared fungal component comprises an ediblemycelium, such as, for example: Hericium erinaceus, Pleurotus ostreatus,Pleurotus eryngii, Pleurotus citrinopileatus, Pleurotus djamor, Trametesversicolor, Lentinula edodes, Armillariella mellea, Tricholomamatsutake, Flammulina velutipes, Volvariella volvacea, Agaricuscampestris, Agaricus blazei, Grifola frondosa, Pholiota nameko, Boletusedulis, Ganoderma lucidum, Ganoderma applanatum, Hypsizygus marmoreus,Morchella hortensis, Morchella angusticeps, Morchella esculenta,Phellinus linteus, Auricularia auricula, Tremella fuciformis, Inonotusobliquus, Fomes fomentarius, Laetiporus sulfureus, Cordyceps sinensis,Cordyceps militaris, Cantharellus cibarius, Polyporus umbellatus, andcombinations thereof. The methods of the invention include screening anumber of strains of fungi and selecting a strain having an enhancedability to grow on, metabolize, or utilize cacao beans or otheragricultural substrate, and/or selecting a strain that is capable ofenhanced removal of one or more undesirable taste components from thecacao beans or other agricultural substrate, and/or enhanced removal ofcaffeine from the cacao beans or other agricultural substrate.

In another embodiment, the prepared fungal component is maintained on anundefined media comprising an aqueous cacao bean, optionally organic, orother agricultural substrate extract and optionally an energy source,or, optionally, the media further comprises an added component such as2-methoxy-3-isopropylpyrazine, catechin, epicatechin, gallic acidequivalents, and/or theobromine The maintenance of the strain of fungicauses an adaptation of the fungi resulting in enhancement of thefungi's ability to grow on, metabolize or utilize cacao beans or otheragricultural substrate, and/or the added component.

The inventive methods also include where the culturing step is afermentation and/or myceliation step carried out under semi-anaerobic oraerobic conditions, where sterile air-flow, relative humidity, lightingand temperature are optionally controlled for, optionally for about 5days.

The methods of the instant invention result in myceliated cacao productshaving reduced levels of undesirable taste components, such as2-methoxy-3-isopropylpyrazine, catechins, theobromine, and increasedlevels of myceliation products, such as fungal (1->3)(1−->6) β-glucans,and other fungal metabolites, relative to the non-myceliated cacaoproducts or other agricultural substrate.

The present invention includes a myceliated cacao product and cacaobeans or other agricultural substrate prepared by the methods of theinvention, and a myceliated cacao or other agricultural product havingreduced levels of undesirable taste components and increased levels ofmyceliation products, such as fungal metabolites, relative to startingor untreated cacao beans or other agricultural substrate.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present invention provides a method for thepreparation of a myceliated cacao (or other agricultural substrate)product. This method includes the step of providing prepared cacao beansor other agricultural substrate, which includes providing the greencacao beans or other agricultural substrate, optionally hydrating theprovided green cacao beans, and sterilizing and/or pasteurizing thecacao beans or other agricultural substrate to provide prepared cacao orother agricultural substrate. The methods of the invention also includethe step of providing a prepared fungal component. The method alsocomprises inoculating the prepared cacao beans or other agriculturalsubstrate in sterile operation with the prepared fungal component andculturing the prepared inoculated cacao beans or other agriculturalsubstrate and prepared fungal component together to effect myceliationto produce the myceliated cacao or other agricultural substrate product.These steps may be performed in any order.

In one embodiment, prepared cacao or other agricultural substrate areprovided, which includes the step of providing cacao or otheragricultural substrate.

One agricultural substrate includes cacao beans or other agriculturalsubstrate. One agricultural substrate is cacao beans. Theobroma cacao,hereinafter “cacao” or “cacao”, belongs to the genus Theobromaclassified under the subfamily Sterculioidea of the mallow familyMalvaceae. There are about 20 different varieties of cacao varieties andhundreds of hybrids and all are suitable for use in the presentinvention, for example, suitable cacao beans are Forastero cacao,Criollo cacao, Trinitario cacao, and Nacional cacao. The presentinvention can be used with any bean from any plant of the genusTheobroma, including any genetically-modified (GMO) strains or cultivarsand also any heirloom variety (non-GMO) strains or cultivars.

Cacao (or cacao) beans suitable for the present invention have beenharvested when the cacao pod is ripe. The average cacao pod produces 20to 50 cacao beans emulsed in mucilaginous pulp. Generally, the cacao podis prepared for the market by fermenting the pod for four to seven days(to remove the pulp) and the beans are dried for another five tofourteen days. The dried beans are suitable for the present invention.

The raw cacao beans have an abundance of naturally occurring bacteriaand other microbes. It is common for cacao beans to be treated withsuperheated steam to mitigate the bacterial content. It is believed thattreating cacao beans with saturated steam also removes some of thedesirable flavor components. Superheated steam-treated cacao beans arealso suitable for the present invention.

Agricultural substrates that may be myceliated in accordance with thisinvention include: all cereals, grains, all species of wheat, rye, brownrice, white rice, red rice, gold rice, wild rice, rice, barley,triticale, rice, sorghum, oats, millets, quinoa, buckwheat, fonio,amaranth, teff and durum; apples and pears, apricots, cherries, almonds,peaches, strawberries, raisins, manioc, cacao, banana, Rubiaceae sp.(coffee), lemons, oranges and grapefruit; tomatoes, potatoes, peppers,eggplant, Allspice, mango powder, Angelica, Anise (Pimpinella anisum),Aniseed myrtle (Syzygium anisatum), Annatto (Bixa orellana), Apple mint(Mentha suaveolens), Artemisia vulgaris, Mugwort, Asafoetida (Ferulaassafoetida), Berberis, Banana, Basil (Ocimum basilicum), Bay leaves,Bistort (Persicaria bistorta), Black cardamom, Black cumin,Blackcurrant, Black limes, Bladder wrack (Fucus vesiculosus), BlueCohosh, Blue-leaved Mallee (Eucalyptus polybractea), Bog Labrador Tea(Rhododendron groenlandicum), Boldo (Peumus boldus), Bolivian Coriander(Porophyllum ruderale), Borage (Borago officinalis), Calamus, Calendula,Calumba (Jateorhiza calumba), Chamomile, Cannabis, Caper (Capparisspinosa), Caraway, Cardamom, Carob Pod, Cassia, Casuarina, Catnip, Cat'sClaw, Catsear, Cayenne pepper, Celastrus paniculatus, Comfrey, Celerysalt, Celery seed, Centaury, Chervil (Anthriscus cerefolium), Chickweed,Chicory, Chile pepper, Chili powder, Cinchona, Chives (Alliumschoenoprasum), Cicely (Myrrhis odorata), Cilantro (see Coriander)(Coriandrum sativum), Cinnamon (and Cassia), Cinnamon Myrtle (Backhousiamyrtifolia), Clary, Cleavers, Clover, Cloves, Coffee, Coltsfoot,Comfrey, Common Rue, Condurango, Coptis, Coriander, Costmary (Tanacetumbalsamita), Couchgrass, Cow Parsley (Anthriscus sylvestris), Cowslip,Cramp Bark (Viburnum opulus), Cress, Cuban Oregano (Plectranthusamboinicus), Cudweed, Cumin, Curry leaf (Murraya koenigii), Damiana(Turnera aphrodisiaca), Dandelion (Taraxacum officinale), Demulcent,Devil's claw (Harpagophytum procumbens), Dill seed, Dill (Anethumgraveolens), Dorrigo Pepper (Tasmannia stipitata), Echinacea,Echinopanax Elatum, Edelweiss, Elderberry, Elderflower, Elecampane,Eleutherococcus senticosus, Epazote (Chenopodium ambrosioides), Ephedra,Eryngium foetidum, Eucalyptus, Fennel (Foeniculum vulgare), Fenugreek,Feverfew, Figwort, Five-spice powder (Chinese), Fo-ti-tieng, Fumitory,Galangal, Garam masala, Garden cress, Garlic chives, Garlic, Ginger(Zingiber officinale), Ginkgo biloba, Ginseng, Ginseng, Siberian(Eleutherococcus senticosus), Goat's Rue (Galega officinalis), Goadamasala, Golden Rod, Golden Seal, Gotu Kola, Grains of paradise(Aframomum melegueta), Grains of Selim (Xylopia aethiopica), Grape seedextract, Green tea, Ground Ivy, Guaco, Gypsywort, Hawthorn (Crataegussanguinea), Hawthorne Tree, Hemp, Herbes de Provence, Hibiscus, Holly,Holy Thistle, Hops, Horehound, Horseradish, Horsetail (Equisetumtelmateia), Hyssop (Hyssopus officinalis), Jalap, Jasmine, Jasmin pearl,Jiaogulan (Gynostemma pentaphyllum), Joe Pye weed (Gravelroot), John theConqueror, Juniper, Kaffir Lime Leaves (Citrus hystrix, C. papedia),Kaala masala, Knotweed, Kokam, Labrador tea, Lady's Bedstraw, Lady'sMantle, Land cress, Lavender (Lavandula spp.), Ledum, Lemon Balm(Melissa officinalis), Lemon basil, Lemongrass (Cymbopogon citratus, C.flexuosus, and other species), Lemon Ironbark (Eucalyptus staigeriana),Lemon mint, Lemon Myrtle (Backhousia citriodora), Lemon Thyme, Lemonverbena (Lippia citriodora), Licorice—adaptogen, Lime Flower, Limnophilaaromatica, Linseed, Liquorice, Long pepper, Lovage (Levisticumofficinale), Luohanguo, Mace, Mahlab, Malabathrum, Manchurian Thorn Tree(Aralia manchurica), Mandrake, Marjoram (Origanum majorana), Marrubiumvulgare, Marsh Labrador Tea, Marshmallow, Mastic, Meadowsweet, Mei Yen,Melegueta pepper (Aframomum melegueta), Mint, Milk thistle (Silybum),Bergamot (Monarda didyma), Motherwort, Mountain Skullcap, Mullein(Verbascum thapsus), Mustard, Mustard seed, Nashia inaguensis, Neem,Nepeta, Nettle, Nigella sativa, Kolanji, Black caraway, Noni, Nutmeg,Mace, Marijuana, Oenothera (Oenothera biennis), Olida (Eucalyptusolida), Oregano (Origanum vulgare, O. heracleoticum), Orris root,Osmorhiza, Olive Leaf (used in tea and as herbal supplement), Panaxquinquefolius, Pandan leaf, Paprika, Parsley (Petroselinum crispum),Passion Flower, Patchouli, Pennyroyal, Pepper (black, white, and green),Peppermint, Peppermint Gum (Eucalyptus dives), Perilla, Plantain,Pomegranate, Ponch phoran, Poppy seed, Primrose (Primula), candiedflowers, dry tea mixes, Psyllium, Purslane, Quassia, Quatre epices,Ramsons, Raspberry, Raspberry (leaves), Reishi, Restharrow, Rhodiolarosea, Riberry (Syzygium luehmannii), Rocket/Arugula, Roman chamomile,Rooibos, Rosehips, Rosemary (Rosmarinus officinalis), Rowan Berries,Rue, Safflower, Saffron, Sage (Salvia officinalis), Saigon Cinnamon, StJohn's Wort, Salad Burnet (Sanguisorba minor or Poterium sanguisorba),Salvia, Sichuan Pepper (Sansho), Sassafras, Savory (Satureja hortensis,S. montana), Schisandra (Schisandra chinensis), Scutellaria costaricana,Senna (herb), Senna obtusifolia, Sesame seed, Sheep Sorrel, Shepherd'sPurse, Sialagogue, Siberian ginseng (Eleutherococcus senticosus),Siraitia grosvenorii (luohanguo), Skullcap, Sloe Berries, Smudge Stick,Sonchus, Sorrel (Rumex spp.), Southernwood, Spearmint, Speedwell,Squill, Star anise, Stevia, Strawberry Leaves, Suma (Pfaffiapaniculata), Sumac, Summer savory, Sutherlandia frutescens, Sweet grass,Sweet cicely (Myrrhis odorata), Sweet woodruff, Szechuan pepper(Xanthoxylum piperitum), Tacamahac, Tamarind, Tandoori masala, Tansy,Tarragon (Artemisia dracunculus), Tea, Teucrium polium, Thai basil,Thistle, Thyme, Toor DaII, Tormentil, Tribulus terrestris, Tulsi (Ocimumtenuiflorum), Turmeric (Curcuma longa), Uva Ursi also known asBearberry, Vanilla (Vanilla planifolia), Vasaka, Vervain, Vetiver,Vietnamese Coriander (Persicaria odorata), Wasabi (Wasabia japonica),Watercress, Wattleseed, Wild ginger, Wild Lettuce, Wild thyme, Wintersavory, Witch Hazel, Wolfberry, Wood Avens, Wood Betony, Woodruff,Wormwood, Yarrow, Yerba Buena, Yerbe mate, Yohimbe, Za'atar, ZedoaryRoot, or derivations thereof in aqueous or semi-aqueous solution(s).

Hydration and Washing Step

In one embodiment, the cacao beans or other agricultural substrate areprepared for use in the instant methods of the invention, resulting in aprepared cacao bean or other agricultural substrate.

In some embodiments, the provided cacao bean or other agriculturalsubstrate is not dried prior to being used in the processes of theinstant invention. In this embodiment, after the cacao bean or otheragricultural substrate is harvested, the cacao bean or otheragricultural substrate optionally has the pulp removed by any processesknown in the art (i.e., the cacao beans can be demucilaged), such asfermentation, or washing and mechanical hulling. Post-demucilaging, thecacao bean or other agricultural substrate can be used in the presentinvention without further treatment, such as drying. The pulp may alsobe retained. Generally, after a fermentation (demucilage) step, thecacao beans have a moisture content of about 55% to 60%. In thisembodiment, the hydration and/or washing step as described below is notnecessary or is obviated by the use of the undried cacao bean or otheragricultural substrate. In some embodiments, the cacao bean or otheragricultural substrate may be partially dried and subsequently hydratedas described herein.

In some embodiments, the cacao beans or other agricultural substrate areprepared by a step of hydrating the cacao beans or other agriculturalsubstrate. Hydration is particularly useful where the cacao beans orother agricultural substrate have been dried. Hydration ensures that thesubstrates have optimal moisture content for the culturing (myceliation)process. Hydration may be accomplished by a number of methods known inthe art.

The hydration may be accomplished by an aqueous medium. The aqueousmedium includes water and optionally, additional excipients. Water maybe, without limitation, filtered, ozonated, deionized, tap, distilled ormineralized. Other excipients can be added to the water, such as buffersto maintain a certain pH, sodium chloride, citric acid and/or ascorbicacid. The pH may be neutral or adjusted. The temperature of the aqueousmedium may be room temperature, or elevated in temperature to acceleratethe hydration process.

Hydration may be accomplished by allowing the cacao beans or otheragricultural substrate to soak in the aqueous medium for any appropriatelength of time, ranging from a few seconds or less to overnight. Cacaobeans, for example, are fairly hygroscopic and will require less time tohydrate. The soaking step for the hydration and/or aqueous extractionstep may be less than a second, at least five seconds, at least tenseconds, at least thirty seconds, at least a minute, at least fiveminutes, at least ten minutes, at least twenty minutes, at least thirtyminutes, at least forty minutes, at least fifty minutes, at least anhour, at least an hour and a half, at least two hours, at least two anda half hours, at least three hours, at least four hours, at least fivehours, at least six hours, at least seven hours, at least eight hours,at least ten hours, at least twelve hours, or at least fifteen hours, atleast eighteen hours, at least twenty four hours, at least thirty sixhours, or at least forty-eight hours. However, the time for thehydration step should be selected in view of the fact that the cacaobeans or other agricultural substrate are not sterile and soaking fortoo long of a time may encourage the growth of undesirable organisms.The hydration time should be selected to optimize the substrate moisturecontent and molecular constituents of the cacao bean or otheragricultural substrates.

The cacao beans or other agricultural substrate may be hydrated at anytemperature that allows for effective hydration; in one embodiment, thetemperature of the aqueous component temperature is room temperature.Hydration temperature should be selected in view of the fact that athigh temperatures, desirable flavor components may be altered.

Hydration may be performed under normal atmospheric pressure or may beperformed under increased pressures to accelerate the hydration processand/or aqueous extraction process, such as between 1 and 2 atmospheres,for example, at 1.5 atmospheres.

Moisture content of the hydrated cacao beans or other agriculturalsubstrate is optionally between about 20% and about 80% moisturecontent, or between a 40% and 70% moisture content. In one embodiment,the moisture content is at least about 30%, at least about 50%, or atleast about 60%. In one embodiment, for cacao bean, the moisture contentis at least about 36%.

The hydration step may occur in a container by any method known in theart. In one embodiment, the container is a drum, such as a 55 gallondrum, or a 5-gallon bucket. In this embodiment, a volumetric 1:1 (equalvolumes) ratio of cacao beans or other agricultural substrate:aqueouscomponent are allowed to stand for 5 minutes to 24 hours. For example,the 55 gallon drum or 5 gallon bucket is filled half-way to the top withcacao beans or other agricultural substrate and aqueous medium is addedto submerge the cacao beans or other agricultural substrate. In thisembodiment, the cacao beans or other agricultural substrate may absorbthe entire aqueous component. In another embodiment, the cacao beans orother agricultural substrate, held in a container, may be filled withwater to completely submerge the beans, in one embodiment, withoutsignificant excess.

In one embodiment, the step of providing prepared cacao beans or otheragricultural substrate optionally includes a step of removingundesirable taste components by washing or rinsing the cacao beans orother agricultural substrate. The wash or rinse may be conducted withthe aqueous medium as described above. In one embodiment, the cacaobeans or other agricultural substrate are optionally washed or rinsedprior to, during, or after the optional hydration step. Washing,draining and/or rinsing the cacao beans or other agricultural substratecan be performed by any method known in the art. The cacao beans orother agricultural substrate may be washed one time, at least two times,at least three times, at least four times, at least five times, at leastten times, at least fifteen times, at least twenty times, at least fiftytimes or more. In one embodiment the wash step is performed two times.The wash or rinse step may include optional soaking times as describedherein.

In another embodiment, water is added to the container holding theprovided cacao beans or other agricultural substrate, and the containerwith the cacao beans or other agricultural substrate is thenheat-treated.

In one embodiment, the cacao beans or other agricultural substrate arewashed by a method of filling a container holding the cacao beans orother agricultural substrate with water to submerge the cacao or otheragricultural substrates, allowing the water to soak for 10 seconds to 4hours, draining the water off and repeating the steps as many times asdesired, or to raise the beans to the desired moisture level. Thewashing or rinsing step may also be carried out until the cacao beans orother agricultural substrate have had a determined amount of undesirabletaste component removed.

The cacao beans or other agricultural substrate may be washed at anytemperature that allows for the efficient extraction of undesirabletaste components; in one embodiment, the temperature of the aqueousmedium temperature is room temperature. Wash temperature should beselected in mind of the fact that at high temperatures, desirable flavorcomponents may be altered, destroyed and/or extracted.

In another embodiment, the excess aqueous medium or component is removedand/or separated and/or drained from the hydrated cacao beans or otheragricultural substrate after the hydration step. This step may also bereferred to as an aqueous extraction step. This step may be done toremove undesirable taste components.

The hydration step, aqueous extraction step, wash and/or rinse step,individually or in combination, can optionally reduce and/or removeundesirable taste components from the cacao beans or other agriculturalsubstrate and may be carried out as described herein until the desiredamount of undesirable taste component has been removed from the cacaobeans or other agricultural substrate.

In some embodiments, undesirable taste components are removed andinclude embodiments where 5% of undesirable taste components areremoved; in other embodiments, up to 10%, up to 15%, up to 20%, up to25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%,up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to90%, or up to 95% of undesirable taste components are removed in theprocesses of the instant invention, including the rinse step. In someembodiments, about 25% to about 80% of the undesirable taste componentsare removed. In one embodiment, about 45 to 50% of the undesirable tastecomponents are removed.

In some embodiments, determination of the extent of the removal of atleast one undesirable taste component is determined by the appearance,taste and/or chemical composition (by methods known in the art) of themyceliated product. Alternatively, the cacao beans' or otheragricultural substrate's appearance or chemical composition may bedetermined by known methods. This determination may be quantitative,e.g., the chemical composition of the myceliated product may be measuredby assay methods, or determined qualitatively by taste testing byskilled persons.

In one embodiment, up to 5% of one or more of the undesirable tastecomponents are removed; in other embodiments, up to 10%, up to 15%, upto 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%,up to 85%, up to 90%, or up to 95% of one or more of the undesirableflavor components are removed in the processes of the instant invention.In one embodiment, one or more of the undesirable flavor components arequantitatively removed.

In one embodiment, reduction of the desirable flavor components such asvolatile oils is minimized by the processes of the present invention. Inprocessing cacao beans or other agricultural substrate, the art teachesto steam treat, steam extract, or stream strip the cacao beans or otheragricultural substrate prior to roasting, which can remove manydesirable volatile oils. The processes of the instant invention avoidthe steam roasting step for, thereby helping to preserve the desirablevolatile oils that contribute to cacao flavor.

Sterilization/Pasteurization

The methods of the present invention further optionally comprise amethod of heat treatment such as pasteurizing and/or sterilizing thecacao beans or other agricultural substrate. In one embodiment, thecacao beans or other agricultural substrate are sterilized to provideprepared cacao beans or other agricultural substrate. This step may beaccomplished by any method known in the art. For example, this step mayperformed under atmospheric pressure or under increased pressure. Thisstep may also be referred to as “pre-processing.” This step is performedto reduce or remove undesirable microbial or fungal organismcontaminants on the cacao beans or other agricultural substrate.

Methods for pasteurization and/or sterilization may be carried out asknown in the art. As an example of pasteurization, cacao beans or otheragricultural substrate may be subjected to dry heat treatment atatmospheric pressure at 145° F. to 190° F. for 30 to 90 minutes, ORalternatively at 140° F. to 210° F. for 20 to 100 minutes.

Sterilization of the cacao beans or other agricultural substrate may beperformed as is known the art. For example, cacao beans or otheragricultural substrate may be sterilized by heating under pressure at 15lb/in² at 121 to 122° C. for 20 to 100 minutes, such as 90 minutes, andadding ¾ lb for every 1,000 ft above sea level. In another embodiment,the steam is superheated to 251 to 255° F. In one embodiment, cacaobeans are sterilized for 80 minutes at 22 psi with slightly drysaturated steam at 255° F. Cacao beans or other agricultural substratemay be sterilized in a container. The container may optionally be thesame container as the container used for the aqueous extraction and/orhydration step. The container may be optionally sealed and the cacaobeans or other agricultural substrate may be sterilized by theapplication of heat to the exterior of the container. In one embodiment,the heat is provided by applying steam to the exterior of the containerfor a sufficient period of time to allow for sterilization of thecontents. In another embodiment, steam is applied to the interior andexterior of the containing for a sufficient period of time to allow forsterilization of the contents.

The sealed container of some embodiments can provide some advantages.For example, sealing the container minimizes outflow of flavorcomponents and aromatic components from the cacao beans or otheragricultural substrate, which can be noticed by the lack of cacao beansor other agricultural substrate aroma from steam from the pressurecooker or autoclave during the sterilization process. Sealing alsoprevents water-soluble flavor and aromatic components from escaping thecacao beans or other agricultural substrate beans directly into steam,hot air, or heated water.

Suitable containers include containers known in the art for mushroomcultivation. Optionally the containers have a section for exchanging airor gases but do not allow passage of any other component. Such sectionsare known in the art and include filter strips. In one embodiment, thecontainer is a food grade fermenter, for example, a 6,000 gallonstainless steel fermenter. In some embodiments, the containers of theinstant invention can be glass, carbon and stainless steel drums,carboys, or polypropylene bags or drums. Fermenters and bioreactors canalso be used as containers of the instant invention. In someembodiments, the containers have a means for gas exchange that precludespassage of contaminants, such as filter zones or valves. In oneembodiment the container is a bag, for example, an autoclavable,polypropylene bag with filter strips, and a gamma-irradiatedpolyethylene bag with filter zones.

A further advantage of the bags described above is that when sealed,they conform to shape of the cacao beans or other agricultural substratebeans when pressurized during the sterilization step. Conforming thebags to the shape of the cacao beans or other agricultural substrateinhibits movement of the cacao beans or other agricultural substraterelative to each other, preventing or minimizing degradation of thesurface of the beans or substrates. This conforming of the bag to theshape of the cacao beans or other agricultural substrate also improvesheat transfer, as the lack of air prevents air insulation of the cacaobeans or other agricultural substrate from heat. The bags can be of anydimension. In one embodiment, bags are elongated or flattened to hastenthe heating process, for example, the length may be three times thediameter of the bag. This dimension may also facilitate the advantageousstacking of bags or positioning of bags for sterilization.

The size of the container or bags to be used can be chosen according tothe volume or amount of cacao beans or other agricultural substrate totreat by the methods of the present invention. Exemplary amounts ofcacao beans or other agricultural substrate to use per bag or containerinclude 1 to 1000 kg of cacao beans or other agricultural substrate,although larger and smaller amounts of cacao beans or other agriculturalsubstrate are contemplated.

In another embodiment, the bags are flattened, having a thickness of1/10th or less than the sum of the peripheral edges of each bag. Thebags can be round in shape, having a circumference that defines theperipheral edges of each bag. Alternatively, the bags can be rectangularso that the sum of the sides defines the peripheral edges of each bag.The bags can be conjoined so that a series of rectangular bags can beeasily handled in a production environment. Bags optionally havebreathable patches (filter strips) that provide for the approximation ofan anaerobic environment. In yet another embodiment, the bags areflattened to hold a layer of beans being less than three beans thick.Accordingly, heat quickly penetrates the flattened bags to the beans toeffectuate sterilization or pasteurization. In this embodiment, due tothe pressurization, the bag will conform to the shape of the cacao beansor other agricultural substrate, and this will yield a pebbled surfaceon the outer surface of each bag when pressure is applied. The pebbledbag surface forms interstitial spaces that allow heat to penetratebetween bags that are stacked to accelerate the sterilization orpasteurization process. The pebbled surface of the bags also inducesturbulent fluid flow along the bag surface to improve heat transfer tothe cacao beans or other agricultural substrate.In another embodiment,the cacao beans or other agricultural substrate are vacuum packed in thebags to eliminate air that could draw volatile flavor or aromaticcomponents from the bags.

In another embodiment, the bags are replaced by sheets of autoclavablematerial, such as BPA-free plastic. One base sheet is continuouslydispensed along the top of a conveyor, cacao beans or other agriculturalsubstrates are then laid on the dispensed base sheet. A second top sheetis overlaid upon the cacao beans or other agricultural substrate andsealed to the base sheet. A vacuum is applied between the top and bottomsheet to evacuate air, then the sheets are sealed at predetermineddistances to form sections. Each section holds a pre-determined volumeof cacao beans or other agricultural substrate. The sections areconveyed through an autoclave, or oven, to effectuate the pasteurizationor sterilization process. Heat may be applied in a pressurized ornon-pressurized environment in the form of steam, hot water underpressure, hot air in turbulent or laminar flow over the sheets, or otherheated fluid. In a variation of this embodiment, the sections containingthe cacao beans or other agricultural substrate are rolled and placed inan autoclave for pressurization or sterilization. One roll can containmany sections.

Since the cacao beans or other agricultural substrate cause a pebbledsurface on the exterior of the sheets, interstitial space exists on theoutside surface of the sheets to hasten the pasteurization orsterilization process by allowing heated fluid to readily penetratebetween sheets. The pebbled sheet surface also induces turbulent fluidflow that further improves heat transfer to the cacao beans or otheragricultural substrate. The pebbled surface inhibits relative movementbetween the beans to assure that the cacao beans or other agriculturalsubstrate do not crack, break or rub.

Fungal Component

The fungal component to use with the present invention can be a fungifrom phylum Basidiomycotina of Eumycota, including any Fungi belongingto Polyporaceae and Hericiaceae, wherein Fungi selected fromBasidiomycotina of Eumycota include Eumycota, including at least oneselected from Basidiomycotina and Ascomycotina, including the strains:Hericium erinaceus, Pleurotus ostreatus, Pleurotus eryngii, Pleurotuscitrinopileatus, Pleurotus djamor, Trametes versicolor, Lentinulaedodes, Armillariella mellea, Tricholoma matsutake, Flammulinavelutipes, Volvariella volvacea, Agaricus campestris, Agaricus blazei,Grifola frondosa, Pholiota nameko, Agrocybe cylindracea, Boletus edulis,Ganoderma lucidum, Ganoderma applanatum, Hypsizygus marmoreus, Morchellahortensis, Morchella angusticeps, Morchella esculenta, Phellinuslinteus, Auricularia auricula, Tremella fuciformis, Inonotus obliquus,Fomes fomentarius, Laetiporus sulfureus, Bridgeoporus nobillismus,Cordyceps sinensis, Cordyceps militaris, Cantharellus cibarius and/or,Polyporus umbellatus, and/or combinations thereof.

Generally, the invention does not contemplate use of the followingfungi: Rhizopus chinensis, R. oligosporus, Aspergillus flavusoryzae, Atamari, A. niger, A. nidulans, A. sojae, Fusarium venenatum, F.graminearum, Saccharomyces cerevisiae, S. exigous, S. pombe,Saccharomycopisis (Candida) lipolytica, Candida utilis, C. krusei or C.tropicalis, Pichia saitoi, Kluyveromyces fragilis, Endomycopsisfibuliger, Chaetomium spp., Zygosaccharomyces rouxii, Mucor racemosus,Geotrichum candidum, Penicillium camemberti, P. notatum, P.griseofulvuum, P. grisea, P. chrysogenum, P. roqueforti, P. nalgiovense,Neurospora intermedia, Amylomyces rouxii, Endomycopsis burtonii,Psycilocibin, Monascus purpureus, Debaryomyces hansenii, Ashbyagossypii, Blakeslea trispora, Tolypocladium niveum, T inflatum,Streptomyces, Neocosmospora, Stachybotrys, Beauveria, Cephalosporiumacremonium, C. acremonium, Gibberella fujikuroi, Fusidium coccineum,Monascus ruber, Claviceps fusiformis, C. paspali, C. purpurea, Amanitamuscaria, or A. phalloides.

Fungal components useful in the present invention may be prepared bymethods as described herein. For example, in one embodiment, a purestrain of fungus is used. In some embodiments, the pure strain of fungusis able to effectively grow on and/or myceliate the prepared cacao beansor other agricultural substrate to prepare the myceliated products. Anystrain of fungus identified herein which is capable of effectivelygrowing on and/or myceliating prepared cacao beans or other agriculturalsubstrate can be used for the methods of the present invention.

Fungal components useful in the present invention may be prepared bymethods as described herein. For example, in one embodiment, a purestrain of fungus is used. It was surprisingly found by the inventors ofthe instant invention that some fungal strains of a particular specieshave enhanced and/or increased ability to grow on, metabolize orotherwise utilize and/or modify cacao beans or other agriculturalsubstrate and/or remove one or more undesirable taste components fromthe cacao beans or other agricultural substrate and/or better toleratethe presence of cacao beans or other agricultural substrate (or extract)in media. In one embodiment, the undesirable taste component ismethylxanthines, such as theobromine and/or2-methoxy-3-isopropylpyrazine. In another embodiment, the fungalcomponent reduces or removes caffeine from cacao beans or otheragricultural substrate.

Therefore, the methods of the invention have as an optional additionalstep a method of selecting a fungal component having an enhanced and/orincreased ability to grow on, metabolize or otherwise utilize and/ormodify cacao beans or other agricultural substrate and/or remove one ormore undesirable taste components from the cacao beans or otheragricultural substrate, and/or remove caffeine and/or better toleratethe presence of cacao beans or other agricultural substrate (or extract)in media. This method comprises screening a number of strains of adesired fungal species to select for a suitable fungal component(strain) which exhibits the enhanced and/or increased ability to growon, metabolize or otherwise utilize and/or modify cacao beans or otheragricultural substrate and/or remove one or more undesirable tastecomponents and/or caffeine from the cacao beans or other agriculturalsubstrate, and using these selected strains in the methods of theinvention.

In one embodiment, a pure strain of any commercially available Ganodermalucidum is used as the fungal component. While all strains of Ganodermalucidum are effective for the present invention, it was surprisinglyfound that some selected strains have the enhanced abilities useful forthe present invention as described herein. One such strain useful forthe fungal component of the present invention is Ganoderma lucidumstrain 806, (Alice Chen; Buffalo, N.Y.; 4/94) commercially availablefrom Pennsylvania State University (The Pennsylvania State UniversityMushroom Culture Collection, available from the College of AgricultureSciences, Department of Plant Pathology and Environmental Microbiology,117 Buckhout Laboratory, The Pennsylvania State University, UniversityPark, Pa., USA 16802). These selected strain(s) were deposited with ATCCas described hereinbelow

This strain was surprisingly determined by the present inventors to moreefficiently grow on, metabolize or otherwise utilize and/or modify cacaobeans or other agricultural substrate and/or remove one or moreundesirable taste components from the cacao beans or other agriculturalsubstrate, and/or better tolerate the presence of cacao beans or otheragricultural substrate (or extract) in media. In another embodiment,this strain can remove and/or reduce the amount of caffeine in cacaobeans or other agricultural substrate. Therefore, in one embodiment, thefungal component is Ganoderma lucidum strain 806 Alice Chen; Buffalo,N.Y.; 4/94. These selected strain(s) were deposited with ATCC asdescribed herein.

In one embodiment, a pure strain of any commercially available Cordycepssinensis is used as the fungal component. While all strains of Cordycepssinensis are effective for the present invention, it was surprisinglyfound that some selected strains have the enhanced abilities useful forthe present invention as described herein. One such strain useful forthe fungal component of the present invention is Cordyceps sinensis(Strain 1009 Caterpillar Fungus; Colorado Corp, 1/2014), commerciallyavailable from Pennsylvania State University (The Pennsylvania StateUniversity Mushroom Culture Collection, available from the College ofAgriculture Sciences, Department of Plant Pathology and EnvironmentalMicrobiology, 117 Buckhout Laboratory, The Pennsylvania StateUniversity, University Park, Pa., USA 16802).

This strain was surprisingly determined by the present inventors to moreefficiently grow on, metabolize or otherwise utilize and/or modify cacaobeans or other agricultural substrate and/or remove one or moreundesirable taste components from the cacao beans or other agriculturalsubstrate, including methylxanthines, such as theobromine and/or2-methoxy-3-isopropylpyrazine and/or better tolerate the presence ofcacao beans or other agricultural substrate (or extract) in media. Inanother embodiment, this strain can remove and/or reduce the amount ofcaffeine in cacao beans or other agricultural substrate. Therefore, inone embodiment, the fungal component is Cordyceps sinensis (Strain 1009Caterpillar Fungus; Colorado Corp, 1/2014). These selected strain(s)were deposited with ATCC as described hereinbelow.

Similarly selected strains for Hericium erinaceus, Pleurotus ostreatus,Pleurotus eryngii, Pleurotus citrinopileatus, Pleurotus djamor, Trametesversicolor, Lentinula edodes, Armillariella mellea, Tricholomamatsutake, Flammulina velutipes, Volvariella volvacea, Agaricuscampestris, Agaricus blazei, Grifola frondosa, Pholiota nameko, Agrocybecylindracea, Boletus edulis, Ganoderma lucidum, Ganoderma applanatum,Hypsizygus marmoreus, Morchella hortensis, Morchella angusticeps,Morchella esculenta, Phellinus linteus, Auricularia auricula, Tremellafuciformis, Inonotus obliquus, Fomes fomentarius, Laetiporus sulfureus,Cordyceps sinensis, Cordyceps militaris, Cantharellus cibarius, and/orPolyporus umbellatus, for example, (or for any species of fungimentioned herein) were obtained by screening a number of strains of eachspecies to select for a suitable fungal component (strain) whichexhibits the enhanced and/or increased ability to grow on, metabolize orotherwise utilize and/or modify cacao beans or other agriculturalsubstrate and/or remove one or more undesirable taste components and/orcaffeine from the cacao beans or other agricultural substrate, and/or isbetter able to tolerate the presence of cacao beans or otheragricultural substrate, and using this selected strain(s) in the methodsof the invention. Therefore, in some embodiments, the selected strain(s)of the fungus identified herein are used in the processes of the instantinvention. These selected strain(s) were deposited with ATCC asdescribed hereinbelow.

Strains mentioned herein are publically available from The PennsylvaniaState University Mushroom Culture Collection, available from the Collegeof Agriculture Sciences, Department of Plant Pathology and EnvironmentalMicrobiology, 117 Buckhout Laboratory, The Pennsylvania StateUniversity, University Park, Pa., USA 16802, and from Fungi Perfecti, POBox 7634, Olympia, Wash. 98507, USA.

All strains referenced herein are deposited with the ATCC at 10801University Boulevard, Manassas, Va. 20110-2209 USA under the BudapestTreaty provisions. The deposit will irrevocably and without restrictionor condition be available to the public upon issuance of a patent andwill be maintained under the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure. These deposits were made merely as aconvenience for those of skill in the art and are not an admission thata deposit is required under 35 U.S.C. §112. However, it should beunderstood that the availability of a deposit does not constitute alicense to practice the subject invention in derogation of patent rightsgranted by government action. The deposit will be maintained withoutrestriction in the ATCC Depository, which is a public depository, for aperiod of 30 years, or 5 years after the most recent request, or for theenforceable life of the patent, whichever is longer, and will bereplaced if it ever becomes nonviable during that period.

Maintenance and Adaptation of Fungal Component

Fungal components useful in the present invention may be prepared bymethods as described herein. For example, in one embodiment, the fungalcomponent is optionally grown, maintained and/or propagated in anundefined medium comprising cacao beans or other agricultural substrateextract prior to use for inoculation of the cacao beans or otheragricultural substrate. In one embodiment, the fungal component isindefinitely maintained in the undefined medium comprising cacao beansor other agricultural substrate extract in the solid-state, floating,and submerged morphologies of various volumes.

Where the agricultural substrate is cacao beans, the extract of cacaobeans may be replaced or spiked by flavor components of the cacao beanwhich are desired to be minimized, metabolized, reduced or removed. Forexample, cacao flavor components may be used, such as2-methoxy-3-isopropylpyrazine and methylxanthines, such as theobromine,obtained commercially as chemical compounds, added to the media as a 0.1M buffer solution through an in-line filtered syringe. The relativeproportions of the flavor components can be adjusted or balanced in themedia to achieve specific flavor profile goals.

Without being bound by theory, the inventors believe that maintenance ofthe fungal component on undefined media comprising cacao beans or otheragricultural substrate plays an important role in the long-termviability and health of the fungal component. It is believed that theperpetual and subtle changes made from batch to batch of agar and liquidmedia when using undefined medium comprising cacao beans or otheragricultural substrate effectively avoids the phenomenon of undesirablegenetic drift that will occur over time to the fungal component when aremaintained on identical (defined) media.

The undefined medium comprising cacao beans or other agriculturalsubstrate extract may be made by a number of methods. In one embodiment,the undefined medium comprises pure aqueous cacao beans or otheragricultural substrate extract. Optionally, additional energy sourcescan be added. Materials are optionally organic and water at least ROfiltered. It has been surprisingly found by the inventors that themedium may comprise aqueous cacao or other agricultural substrateextract without any additional added excipients, such as an additionalenergy source for growing fungi of the present invention.

Solid media comprising undefined medium comprising cacao beans or otheragricultural substrate extract. In one embodiment, undefined mediumcomprising cacao beans or other agricultural substrate agar media togrow fungi for the eventual purpose of myceliating sterilized cacaobeans or other agricultural substrate can comprise filtrate of cacaobeans or other agricultural substrate. In one embodiment, 0.1 to 100lbs. of cacao beans or other agricultural substrate are in 0.1 to 100 Lof water, respectively, for 1 minute to 12 hours. The filtrate wascollected through 1 to 3 filtrations of the mixture, and 14 to 17 g/L ofagar was added. The base extract solution can be used alone, or co-mixedwith vegetable extracts and/or additional sugar sources such as fruitjuice.

This base solution can be optionally mixed with the filtrate ofundefined vegetable (of any type but ideally organic) aqueous extract,such as malt extract, yeast extract, potato, etc. In one embodiment thevegetable is potato. Aqueous potato mixture can be prepared by softening1 to 300 g of potato mass in boiling or pressurized water, mashed, andthe filtrate was collected through 1 to 3 filtrations. Optionally, fruitjuice with no added sugars can also be added to the base cacao beans orother agricultural substrate agar media. In one embodiment, the mediumcomprises 0.1 to 10% by weight of malt extract, 0.1 to 10% by weightundefined vegetable extract with essence of cacao bean, 0.1 to 10% byweight of yeast extract, 0.1 to 10% by weight of peptone, 0.1 to 10% byweight of glucose, 20 to 80% by weight of water, and 1 to 90% by weightwhole cacao beans or other agricultural substrate or green coffee beanextract.

As a non-limiting example of the media, for example, 2 lb cacao beans orother agricultural substrate, either pulverized or whole can be mixedwith ¼ gallon water at room temperature. The mixture may be blended. Themixture is then allowed to extract for 20 minutes with shaking, thenfiltered three times through fine mesh. This solution can be used alone,or mixed with the following: about 5 organic potatoes are placed in 10 Lof water and autoclaved 20 minutes to soften the potatoes. The potatoesare then pulverized with a potato masher, and then filtered through finemesh three times. 1 L of commercial unsweetened fruit juice can beadded. The potato extract is added to the cocoa extract and autoclaved.

In another embodiment, the media may comprise a chocolate liquor, whichcan be made by methods known in the art, such as obtaining a fine groundwhich is a liquid. The liquor can be used neat or can be diluted asdesired. A chocolate liquor media should be agitated.

Once prepared, the media can be sterilized by any method known in theart. Once the media is cooled, can be poured into Petri plates andfungal cultures were propagated from plate to plate in sterileoperation, as known in the art. Slants for test tubes and flasks may beprepared by this method. Petri plates can be inoculated with floatingand submerged liquid tissue culture, and with myceliated substrate.

Liquid media undefined medium comprising cacao beans or otheragricultural substrate extract. Cacao beans or other agriculturalsubstrate extract, and undefined vegetable extract were prepared asdescribed for solid media, except that no agar is added. If preparing tomake a floating culture, 1 to 10 tablespoons of flour was added to themixture, in one embodiment, about 1 tablespoon per 10 to 15 L ofculture. The media can be sterilized by methods known in the art. Oncecooled, the vessel can be inoculated in sterile operation with acolonized section petri plate, from other liquid tissue cultures, orfrom samples of myceliated substrate.

In one embodiment, the fungal component for inoculation into cacao beansor other agricultural substrate can be prepared by submerged liquidtissue culture using the undefined medium comprising cacao beans orother agricultural substrate extract liquid media as defined herein andagitated on a shaker table. In one embodiment, the agitation rate is 50to 240 rpm, or 85 to 95 RPM, and incubated for 4 to 90 days. In oneembodiment, the temperature of incubation is 87 to 89° F.

In one embodiment, the fungal component is trained and/or adapted and/ormaintained in its ability to efficiently grow on, metabolize orotherwise utilize and/or modify cacao beans or other agriculturalsubstrate. In one embodiment, the fungal component is selected and/ortrained and/or adapted and/or maintained in its ability to remove orreduce one or more undesirable taste components from the cacao beans orother agricultural substrate or to remove or reduce the amount ofcaffeine. Methods to determine whether an undesirable taste componentand/or caffeine has been reduced or removed has been disclosed hereinand also be found in the art.

In one embodiment, the trained and/or adapted and/or maintained fungalcomponent is prepared from disinfected wild and healthy fungi. Suchfungi with changed, improved, and adapted properties as describedherein, relative to the starting strains, either selected or unselected,were developed by these methods. These adapted strains were depositedwith the ATCC as described elsewhere herein. In one embodiment, thetrained and/or adapted and/or maintained fungal component is preparedfrom Ganoderma lucidum. In one embodiment, the trained and/or adaptedand/or maintained fungal component is prepared from Ganoderma lucidumstrain 806 Alice Chen; Buffalo, N.Y.; 4/94. In another embodiment, thetrained and/or adapted and/or maintained fungal component is preparedfrom Cordyceps sinensis (Strain 1009 Caterpillar Fungus; Colorado Corp,1/2014). In one embodiment, the trained and/or adapted and/or maintainedfungal component is prepared from Hericium erinaceus, Pleurotusostreatus, Pleurotus eryngii, Pleurotus citrinopileatus, Pleurotusdjamor, Trametes versicolor, Lentinula edodes, Armillariella mellea,Tricholoma matsutake, Flammulina velutipes, Volvariella volvacea,Agaricus campestris, Agaricus blazei, Grifola frondosa, Pholiota nameko,Agrocybe cylindracea, Boletus edulis, Ganoderma lucidum, Ganodermaapplanatum, Hypsizygus marmoreus, Morchella hortensis, Morchellaangusticeps, Morchella esculenta, Phellinus linteus, Auriculariaauricula, Tremella fuciformis, Inonotus obliquus, Fomes fomentarius,Laetiporus sulfureus, Cordyceps sinensis, Cordyceps militaris,Cantharellus cibarius, and/or Polyporus umbellatus. These fungi havingchanged, improved, and adapted properties as described herein, relativeto the starting strains, were deposited with ATCC as described herein.

The training and/or adaption and/or maintenance step as described hereincan be optionally conducted on undefined medium comprising cacao beansor other agricultural substrate extract liquid media or solid media asdefined herein. In one embodiment, the fungi may be cultivated for 4 to90 days at any temperature known in the art for cultivating fungi, forexample, 87 to 89° F. Re-inoculation of the cultivated fungal componentinto fresh media as described herein can be performed at an appropriatetime as determined by one of skill in the art depending on the growthrate, growth cycle, and appearance of the fungal component. The cycle ofgrowth and re-inoculation of the fungal component into fresh media, insome embodiments, is performed more than one time, more than two times,more than three times, more than four times, more than five times, morethan ten times, more than fifteen times, more than twenty times, morethan twenty five times, more than thirty times, more than forty times,more than fifty times, more than seventy five times, or one hundredtimes or more. The fungal component by these methods can, for example,better recognize cacao beans or other agricultural substrate or anyparticular component of cacao beans or other agricultural substrate asan energy source, better tolerate the presence of cacao beans or otheragricultural substrate extract in media (as measured by an enhancedgrowth rate, for example), better remove undesirable taste components,or better remove caffeine. In one embodiment, the undesirable tastecomponent to be removed and/or reduced are catechins, methylxanthines,such as theobromine or 2-methoxy-3-isopropylpyrazine.

Therefore, the methods of the invention have as an optional additionalstep, a method of preparing a trained and/or adapted and/or maintainedfungal component comprising a fungal component having an enhanced and/orincreased ability to grow on, metabolize or otherwise utilize and/ormodify cacao beans or other agricultural substrate and/or remove one ormore undesirable taste components from the cacao beans or otheragricultural substrate, and/or remove caffeine. Thus use of the trainedand/or adapted and/or maintained fungal component is contemplated forthe present invention. The methods of the invention further comprise useof any of the trained, adapted, and/or maintained fungal component(s) asdescribed herein, in the methods of the instant invention.

Preparation of Fungal Component for Inoculation of Cacao Beans or OtherAgricultural Substrate

In one embodiment, methods for preparing the fungal component toinoculate the prepared cacao beans or other agricultural substrateinclude scaling up a fungal component as defined herein in liquidculture. Such fungal component which is readied for inoculation of theprepared cacao beans or other agricultural substrate is called a“prepared fungal component.”

In one embodiment, the prepared fungal component is in solid culture. Inanother embodiment, the prepared fungal component is in liquid culture.In another embodiment the prepared fungal component is a mixture ofsolid and liquid culture. Liquid culture can be accomplished by anymeans known in the art and includes use of a bioreactor. For example,when using a bioreactor to prepare the fungal component, the bioreactorcan be prepared by diluting undefined medium comprising cacao beans orother agricultural substrate extract liquid media up to 1000× withfiltered/RO water. The jacket of the bioreactor may be steamed in oneembodiment to sterilize the media, or alternatively, the media can besterilized by way of injecting steam into the vessel.

The media to use in preparation of a fungal component for use toinoculate the prepared cacao beans or other agricultural substrate, maybe any suitable media known in the art, or may be made by the methodsdisclosed herein. The media may further comprise trace elements andorganic substances such as water, nucleic acids, and minerals. The mediamay be diluted up to 1000× with filtered/RO water. Dilution can be 1×,about 2×, about 3×, about 4×, about 5×, about 6×, about 7×, about 8×,about 9 x, about 10×, about 15×, about 20×, about 25×, about 30×, about35×, about 40×, about 45×, about 50×, about 55×, about 60×, about 65×,about 70×, about 80×, about 90×, about 100×, about 150×, about 200×,about 250×, about 300×, about 350×, about 400×, about 450×, about 500×,about 550×, about 600×, about 650×, about 700×, about 750×, about 800×,about 850×, about 900×, about 950× or about 1000×. In some embodiments,the dilution is about 5× to about 100×. For a 100 L bioreactor, mediacan be diluted about 10×, for example.

In one embodiment, to inoculate the reactor, media may be pumped fromanother reactor through a sterilized line with an inline pump, orcreating positive pressure by sparging air into the reactor with an aircompressor that runs the air through inline 0.2/0.5 micron capsulefilters then through a check valve with a specific cracking pressure,for example, 2 to 3 psi.

Methods to inoculate the bioreactor to prepare the fungal componentinclude inoculating the bioreactor with an excised colonized section ofPetri plate and/or a sample of liquid culture using sterile procedure,or by pouring a floating or submerged liquid tissue culture into thebioreactor through the nozzle. Bioreactors can be inoculated in anon-clean space if they are connected to a valved off containercontaining a prepared glycerol stock, the container being connected to asteamable line that can be sterilized, cooled, and re-pressurized withsterile air before opening the line between the container andbioreactor.

Optionally, the fungal component may be agitated during culturing bymethods known in the art. For example, in a bioreactor, the agitationmay be accomplished by a combination of sparged air and a motorizedpaddle which allows both a turbulent environment and shear mechanicalforce. The inventors, without limitation, have found that thecombination is superior to running either method individually, assparged air creates the most turbulence at the top half of the culture,while affecting the bottom less, which can be kept agitated by amotorized paddle, while the paddle does not have to run at such a highRPM as normally used in the art. The combination creates the propersmall hyphal sphere sizes without damaging the mycelia.

Liquid state fermentation agitation and swirling techniques are known inthe art and include mechanical shearing using magnetic stir bars,stainless steel impellers, injection of sterile high pressure ambientair, injection at high-pressure of sterile media, and or the use ofshaker tables. Higher agitation and swirling rates, in conjunction withair and media injections, produce small mycelial spheres.

The fungal component can be grown until ready for inoculation of theprepared cacao beans or other agricultural substrate as determined byone of skill in the art. In some embodiments, the fungal component canbe grown for 48 hours prior to use in inoculating the cacao beans orother agricultural substrate. Determination of whether the fungalcultures comprising the fungal component are suitable for inoculation ofthe prepared cacao beans or other agricultural substrate can bedetermined by one of skill in the art. For example, in one embodiment,the fungal culture, when in liquid media, is suitable for inoculationwhile in exponential growth phase, either early or late. Senescentcultures and cultures in earlier growth phases with lower amounts ofmycelia/mL can be used, but are not preferred. The prepared fungalcomponent optionally appears well grown through in the media, withvisible mycelia growing through every mL visible by microscope andunassisted vision.

In order to effect the most efficient myceliation of the cacao beans orother agricultural substrate, the fungal component have defined hyphalsphere sizes which enables hyphae growth in three dimensions around thespherical conglomeration of the culture of the fungal strain. In oneembodiment the hyphal sphere size is less than 10 mm in diameter, lessthan 2 mm in diameter, less than 1 mm in diameter, less than 100 μm indiameter, less 10 μm in diameter, less than 5 μm in diameter, less than2 μm in diameter, or less than 1 μm in diameter. In another embodiment,the hyphal spherical conglomeration has a size range of 5 μm to 1 μm indiameter, or, a size range of 10-50 μm in diameter.

These methods result in a prepared fungal component for inoculation ofprepared cacao beans or other agricultural substrate.

Inoculation and Myceliation of the Prepared Cacao Beans or OtherAgricultural Substrate

The prepared cacao beans or other agricultural substrate are inoculatedwith the prepared fungal component. The prepared fungal component to beused can be any fungal component as defined in the instant invention.The inoculation of the prepared fungal component onto the prepared cacaobeans or other agricultural substrate can be carried out by any methodknown in the art. This step may be variously referred to as theculturing step, the fermentation step, and/or the myceliation step.

The myceliation may take place in a container as described herein. Inone embodiment, the myceliation takes place in a 55 gallon drum asdescribed herein. In this embodiment, the 55 gallon drums has a lidcontaining two ports, and one port can be used as an inoculation port,while the other can be used to sparge in filtered air to the bottom ofthe culture, and another port to serve as a vent. In some embodiments,the inoculation port is a quick disconnect socket, which is attached toa quick disconnect plug at the end of a harvesting line duringinoculation. Optionally, prepared cacao beans or other agriculturalsubstrate in a plurality of drums can be myceliated in one cycle via asterilized manifold connected to bioreactor harvesting line, with theincluded infrastructure to target any drum in singular or all togetherat once. In one embodiment, a system for volumetrically consistentdispensing of inoculant per culture is used.

In one embodiment, the culture may be pneumatically injected into acontainer comprising the prepared cacao beans or other agriculturalsubstrate. Moisture may optionally be injected into bags to optimizemycelial growth. In another embodiment the cacao beans or otheragricultural substrate are inoculated by pouring the culture into thecontainer holding the sterilized cacao beans or other agriculturalsubstrate either manually or through a valve built into the fermenter orbioreactor, from any variety of liquid tissue culture.

In one embodiment, the prepared cacao beans or other agriculturalsubstrate are cooled to a temperature of between 80 to 100° F. prior toinoculation with the prepared fungal component. Cooling may beaccomplished by refrigeration or at room temperature. The step ofmyceliating the prepared cacao beans or other agricultural substrate cantake place for between 4 and 90 days, for between about seven and twentyone days, and in one embodiment, for about seven days, and at anytemperature, for example, at 87 to 89° F. Multiplication of the fungalmycelia by fermentation is carried out by efficiently controllingenvironmental light, such as by a control model of 40% lighting and 60%dark, and also by controlling sterile airflow and temperature at 86 to88 or 87 to 89° F., or between 12 to 35° C., or between 24° C. to 32° C.

Relative humidity of this culturing, myceliation and/or fermentation isbetween 20% and 80%, in some embodiments, at least about 60%. In someembodiments, the relative humidity is at least about 36 and 40%.

In an alternate embodiment, prior to addition of the fungal component,the cacao beans are prepared for liquid myceliation (fermentation)culture. The cacao beans are ground into fine particles, preferably,less than 1 mm in diameter as approximate particle size. In oneembodiment, the particles are 20 to 50 μm in size. The cacao beans arethen placed into aqueous solution that has a viscosity regulated oradjusted to suspend the cacao particles. Preferably, the aqueoussolution has a viscosity adapted to the cacao particle size to enablethe cacao particles to be suspended in the aqueous solution whenagitated. The aqueous media containing the fungal component and thecacao particles can be deposited in a bioreactor of any size. Preferablythe bioreactor is sized to hold at least 10 liters of aqueous media.More preferably, the bio-reactor is sized to hold several thousandliters, or more.

In one embodiment, the liquid myceliation culture is agitated. Theagitation means is regulated to optimize the aqueous solution tomaintain the mass of fungal mycelium in small particles. Preferably thefungal mycelium will have hyphal spheres, or conglomerations, havingsizes that are between 1 to 100 μm in diameter. In another embodiment,the hyphal conglomerations are smaller than the particle size of thecacao.

In liquid myceliation culture, the bio-reactor is maintained at asuitable temperature and pressure to enable the mycelium to digestcomponents of the cacao in a facultative anaerobic environment. Afterthe mycelium has digested components of the cacao to remove bitterflavor components, the cacao is separated from the aqueous solution.This can be done by ceasing agitation and allowing the cacao toprecipitate. This can also be accomplished by filtering the cacao fromthe aqueous solution. After the cacao is removed from the aqueoussolution, it may be prepared for processing (including drying the cacaointo a powder). A secondary step is optionally performed to extract theaqueous solution after removal of the cacao. This extract can be used torecover water-soluble flavor components of the cacao. Thesewater-soluble flavor components may be processed and may be added backto the cacao either as a powder or an aqueous spray or bath. Cacaopowder is the result. Supplemental flavor is added, as needed, tofurther improve the flavor of the cacao. For example sweeteners or cacaoflavor extracts may be added at this juncture in the process.

Accordingly, the cacao is ready to form into any cacao product, forexample, chocolate.

The step of myceliating the prepared cacao beans or other agriculturalsubstrate is preferably accomplished in an aerobic, anaerobic orsemi-anaerobic environment. Methods known in the art can be used tooptionally induce and/or maintain facultative anaerobic metabolicactivity of the prepared fungal component as described by the Pasteureffect. In an alternate embodiment, the cacao beans or otheragricultural substrate are deposited in large stainless steel fermentersin a non-sterile environment. The fermenters regulate sterile-airconcentration, humidity, and temperature, and optionally enable thefacultative anaerobic activity and mycelial growth on the prepared cacaobeans or other agricultural substrate. Facultative anaerobic activitymetabolizes more cellulose of per unit of time, meaning that the cacaobeans or other agricultural substrate substrate is consumed at a morerapid rate than in an aerobic environment. In some cases mycelial growthis nine times faster than in an aerobic environment (that is, nine timesmore cellulose molecules are metabolized to ATP). Another benefit isthat the anaerobic environment inhibits fruiting body growth. Ananaerobic environment also assures a reduction in unwanted bacterialgrowth, and other unwanted microbial growth.

Expansion of the fungus mycelia is monitored by microscopy, andschedules of growth documented by photography.

The longer the incubation period, the greater the production of themycelium dry weight and the greater the flavor enhancement of the cacaobeans or other agricultural substrate. Some strains will form primordialtissue and fruit bodies by 30 days (Hericium erinaceus is particularlyprone to fruiting while in culture, as is Ganoderma lucidum andFlammulina velutipes). In some embodiments, harvesting the myceliatedcacao beans or other agricultural substrate beans is performed beforethe culture fruits body tissue. However, a prolonged period ofincubation of a specific fungus does not guarantee a high production ofmetabolite or the accumulation of mycelia and/or myceliation products.

Determination of when to harvest the myceliated product may bedetermined by a number of methods. Harvesting is generally performedwith a timing to optimize the taste profile of the myceliated productaccording to the taste profile desired. For example, the scent profileof the myceliation culture can be used by the trained person todetermine when the culture is ready. Determination of the appearance ofthe culture may also be done by the trained person. In some embodiments,harvesting can be done when the amount of the mycelia in the culture arein the approximate amount of 2 to 3 fully grown (standard size) Petriplates (for G. lucidum), or when the amount of the mycelia are in theapproximate amounts of 10 to 12 fully grown standard Petri plates (forC. sinensis), per 8 lb of cacao beans or other agricultural substrate.Analytical methods of analysis including high performance liquidchromatography, mass spectroscopy, and/or NMR may be employed to carryout measurement of total biomolecules in order to determine the optimumcomposition and cultivation conditions and the appropriate time(s) forharvesting the fungi.

In a non-limiting example of the present invention, about 8 lb ofprepared cacao beans or other agricultural substrate according to theprocesses of the invention, in a sealed container as described herein,was inoculated with about 400 ml of prepared I. obliquus fungalcomponent as described herein. An aerobic environment was obtained. Themyceliation or culturing was allowed to proceed for seven days at 87 to89° F., although as little as four days or as long as sixty or more daysis also suitable. Harvesting was performed when observers determinedthat an appropriate taste profile for the myceliated product had beenobtained.

Reduction of Undesirable Taste Components During Myceliation

Cacao beans and other agricultural substrates may contain undesirabletaste components. Such undesirable taste components may be defined orundefined. In one embodiment, undesirable taste components include, forexample, catechin, epicatechin, gallic acid equivalents,2-methoxy-3-isopropylpyrazine, and methylxanthines such as theobromineOne embodiment of an undesirable taste component includes2-methoxy-3-isopropylpyrazine. This component is metabolized by thefungal component into polysaccharides, such as fungal (1−>3)(1−>6)β-glucan. Other undesirable taste components include bitter tastecomponents. One such bitter component is methylxanthines, such astheobromine, and catechin. Myceliating raw cacao yields a cacao beanthat is less bitter that raw cacao that is not myceliated.

Myceliation of cacao also causes the shell of the bean to be more easilywinnowed for advantages in processing the cacao beans. It also resultsin a chocolate product that is much less prone to blooming

The step of culturing or myceliation may also cause reduction and/orremoval of undesirable taste components as described herein and/orcaffeine. In some embodiments, determination of the extent of theremoval of at least one undesirable taste component is determined by theappearance, taste and/or chemical composition of the myceliated productas is known in the art. This determination may be quantitative, e.g.,the chemical composition of the myceliated product may be measured byassay methods for one or more of the undesirable taste components byassay methods as known in the art, or determined qualitatively by tastetesting by skilled persons.

In one embodiment, up to 5% of one or more of the undesirable tastecomponents are removed; in other embodiments, up to 10%, up to 15%, upto 20%, up to 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%,up to 85%, up to 90%, up to 95%, or up to 99.99% of one or more of theundesirable flavor components are removed in the processes of theinstant invention. In one embodiment, one or more of the undesirableflavor components are quantitatively removed. The invention also relatesto myceliated products having reduced levels of undesirable tastecomponents as described herein.

In one embodiment, up to 10%, up to 15%, up to 20%, up to 25%, up to30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%,up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, or upto 95% of undesirable taste components are removed in the processes ofthe instant invention.

In one embodiment, the undesirable taste component is2-methoxy-3-isopropylpyrazine, and up to 5% of2-methoxy-3-isopropylpyrazine is removed; in other embodiments, up to10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%,up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to75%, up to 80%, up to 85%, up to 90%, or up to 95% of2-methoxy-3-isopropylpyrazine is removed in the processes of the instantinvention. The invention also relates to myceliated cacao productshaving reduced levels of 2-methoxy-3-isopropylpyrazine as describedherein.

In one embodiment, the undesirable taste component are methylxanthinessuch as theobromine, and up to 5% of methylxanthines such as theobromineare removed; in other embodiments, up to 10%, up to 15%, up to 20%, upto 25%, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%,up to 90%, or up to 95% of methylxanthines such as theobromine areremoved in the processes of the instant invention. The invention alsorelates to myceliated cacao products having reduced levels ofmethylxanthines such as theobromine as described herein.

In one embodiment, caffeine is removed from the cacao beans or otheragricultural substrate during the culturing or myceliation step. In oneembodiment, up to 5% of caffeine is removed; in other embodiments, up to10%, up to 15%, up to 20%, up to 25%, up to 30%, up to 35%, up to 40%,up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to75%, up to 80%, up to 85%, up to 90%, or up to 95% of caffeine isremoved in the processes of the instant invention. The invention alsorelates to myceliated products having reduced levels of caffeine asdescribed herein.

Removal of undesirable taste components may allow for increasing thevalue of poorer quality cacao or other agricultural products and/orrendering it more edible. Myceliated products produced by this methodmay be used to blend with less expensive cacao beans or otheragricultural substrate leading to a lower cost product having improvedtaste properties. The amount of sugar, milk and substitutes thereof tobe added to the myceliated products may be reduced. The instant methodslead to enhanced flavor profile of the myceliated products due to aperception that the myceliated products provide a richer, smoother,and/or sweeter food with less bitter, harsh, and/or acidic tastes.

Addition of Flavor and/or Health Promoting Components

The culturing or myceliation processes of the instant invention, in someembodiments, provide a myceliated cacao beans or other agriculturalsubstrate product with added flavor and/or heath promoting components.For example, the myceliated cacao beans or other agricultural substrateproducts may contain exogenously added anti-tumor and immunomodulatoryhealth promoting components.

Fungi are metabolically similar to animals but structurally similar toplants in that they possess a rigid cell wall formed largely of longsugar molecule chains joined by somewhat difficult to digest beta (β-)linkages and to a smaller extent more easily digestible alpha (α-)linkages in conjunction with membrane-bound proteins. In contrast, plantcell walls (such as cacao beans or other agricultural substrate) aremade of cellulose polysaccharides whose (1−>4) β-glycosidic glucoselinkages are non-digestable by humans, but are digestable by fungi.Fungi cell walls are primarily composed of (1−>3) β-glycosidic linkages,with (1−>6) linked side chains, and chitin, and therefore may be brokendown by minimal processing using water, heat and mechanical treatmentinto smaller, more easily digestible, immunologically-activepolysaccharide molecules of variable microparticulate size calledβ-glucans, and related glycoprotein compounds. The immune response toβ-glucan is dependent upon α- or β-glucan side chain structure, whichhas primary, secondary, and chiral tertiary structures, explaining thedifferences in immune response to each fungi's unique α- and or β-glucanprofile. Thus, myceliated cacao beans or other agricultural substrateproducts have added health promoting components including the moleculesdescribed above. Other health promoting components present in themyceliated cacao beans or other agricultural substrate products may becomponents that have various properties such as immunomodulating,anti-aging, aphrodisiac, anti-tumour, anti-viral, anti-bacterial, and oranti-fungal properties and include compounds such as α- and β-glucans,glycoproteins, proteins, ergosterols, sterols, triterpenes, and fattyacids, glucomannan, riboglucan, sterpuric acid, mannitol, ribitol,guanosine and adenosine.

Methyl pyrazines, which develop naturally in cacao, are among the mostimportant desirable flavor compounds found in cacao. In the presence ofheat, such as roasting, they are produced through Maillard reactionsi.e. between sugars and amino acids or peptides. Without being bound bytheory, it is believed that methyl pyrazines are also be producedthrough fermentation and/or myceliation, which leads to increased and/oraltered methyl pyrazines in myceliated cacao beans and otheragricultural products. Using this method of manufacture of cacao, asingle strain of fungi decomposes the cellulose, alkaloid, fat, andprotein content contained in the prepared cacao beans and changes thesubstance structure and concentration of the cacao beans. On the otherhand, a uniform and consistent quality of cacao is obtained by thisinvention's method of fermentation for processing raw cacao beans. Byutilizing methods disclosed herein induced by this clean fermentationprocess, the flavor of raw cacao beans is enhanced, making the resultingcacao much richer, smoother, sweeter, and unique (compared to plaincacao made from non-myceliated beans).

The reactive carbonyl group of the sugar reacts with the nucleophilicamino group of the amino acid, and forms a complex mixture of poorlycharacterized molecules responsible for a range of odors and flavors.This process is accelerated in an alkaline environment (e.g., lyeapplied to darken pretzels), as the amino groups are deprotonated and,hence, have an increased nucleophilicity. The type of the amino aciddetermines the resulting flavor. This reaction is the basis of theflavoring industry. At high temperatures, acrylamide can be formed.

Agaricus blazei may be used for addition of unique α- and β-linkedglucans called glucomannan and riboglucan, which are antiviral, into themyceliated cacao beans or other agricultural substrate product. Other A.blazei polysaccharide extracts may have anti-cancer effects and may beco-therapeutic with other mycelial extract of Fungi listed herein.Methods to optimize biomass and extracellular polysaccharide productionhave been reported. Therefore, myceliation with A. blazei and myceliatedcacao beans or other agricultural substrate products containing flavorand/or health promoting components derived from A. blazei as describedherein are also included in the instant invention.

Cordyceps sinensis produces cordycepic acid, adenosine, D-mannitol, andcordycepinadenosine which are immunomodulating and anti-viral. C.Sinensis extracts have been shown to be anti-aging and aphrodisiacal.Mycelial sterols isolated from C. sinensis have been shown to inhibitthe proliferation of numerous cancer cell lines. C. sinensis mycelialpolysaccharide extracts have been shown to induce hypoglycemia.Therefore, myceliation with C. sinensis and myceliated cacao beans orother agricultural substrate products containing flavor and/or healthpromoting components derived from C. sinensis as described herein arealso included in the instant invention.

Flammulina velutipes mycelium has been shown to have a polysaccharideprofile that is immunomodulating. F. velutipes mycelium composes aunique ergosterol and amino acid profile, sterpuric acid, mannitol,ribitol, and the nucleosides guanosine and adenosine, Enokipodins A-Dextracted from F. velutipes mycelium are broad spectrum antimicrobialterpenes. The proteins flammulin and velutin exhibit anti-HIV andanti-HPV activity. Therefore, myceliation with F. velutipes andmyceliated cacao beans or other agricultural substrate productscontaining flavor and/or health promoting components derived from F.velutipes as described herein are also included in the instantinvention.

Ganoderma lucidum's polysaccharide profile has been shown to beimmunomodulating in human cell lines and also in clinical studies. G.lucidum mycelial extracts have anti-peroxidative, anti-inflammatory, andanti-mutagenic properties. G. lucidum extracts have been shown to beanti-aging and aphrodisiacal. The triterpenoid profile of G. lucidum hasbeen determined and shown to be anti-hepatotoxic and hepatoprotective,anti-tumor, anti-angiogenic, anti-hypertensive, hypocholesterolemic,anti-histaminic, and anti-HIV. G. lucidum, in addition to producingpolysaccharides and glycoproteins, likewise produce triterpenes, such asganoderic and lucidenic acids, phenolic compounds, and sterols whichalso have high biological activity and therapeutic properties and are inthemselves anti-oxidant, anti-tumor, anti-bacterial, anti-cancer,anti-inflammatory, anti-histamine, hypotensive, sedative, and meditativeafter oral consumption. Therefore, myceliation with G. lucidum andmyceliated cacao beans or other agricultural substrate productscontaining flavor and/or health promoting components derived from G.lucidum as described herein are also included in the instant invention.

Grifola frondosa's polysaccharide profile has been shown to beimmunomodulating and anti-oxidative, G. frondosa produces ergosterolsand an anti-oxidative profile of fatty acids. The anti-tumor effects ofG. frondosa extracts on in vitro cancer cell lines have beeninvestigated, and shows promise for diabetes patients as beinghypoglycemic. Therefore, myceliation with G. frondosa and myceliatedcacao beans or other agricultural substrate products containing flavorand/or health promoting components derived from G. frondosa as describedherein are also included in the instant invention.

Hericium erinaceus mycelial and fruiting body extracts have been shownto be anti-mutagenic and immunomodulatory across various cell lines. H.erinaceus uniquely produces hericenones in fruit bodies and erinacinesin mycelium, structurally determined compounds that can pass theblood-brain bather and promote secretion of Nerve Growth Factor (NGF) incertain regions of the brain. Erinacenes have been shown to be greaterpotentiators of NGF expression than hericenones. Therefore, myceliationwith H. erinaceus and myceliated cacao beans or other agriculturalsubstrate products containing flavor and/or health promoting componentsderived from H. erinaceus as described herein are also included in theinstant invention.

Aspects of Lentinula edodes' polysaccharide profile has been determinedand shown to be immunomodulating and antiviral. Lentinan and othermetabolites have been studied for their numerous health care benefits.In some countries, lentinan is classified as an “antineoplasticpolysaccharide” and is available for clinical use. Addition of lentinanto standard cancer therapies has been shown to result in increased tumornecrosis and with hepatocellular carcinoma and improved quality of lifein patients with esophageal carcinoma. Therefore, myceliation with L.edodes and myceliated cacao beans or other agricultural substrateproducts containing flavor and/or health promoting components derivedfrom L. edodes as described herein are also included in the instantinvention.

Phellenis linteus extracts have been shown to exhibit antitumoractivity. Polyporus umbellatus polysaccharide extracts have been studiedand shown to be anti-cancer, immunomodulating, anti-malarial, andhepatoprotective. Inonotus obliquus mycelial polysaccharide extract hasdemonstrated anti-tumor, hypoglycemic, and anti-oxidative properties.Pleurotus ostreatus mycelium and fruit body composition have been shownto be very similar, differing only in amino acid content. The mycelialpolysaccharide profile consists primarily of laminarin, the extract ofwhich has been shown to be immunomodulating. Lovastatin, isolated fromthe mycelial broth of P. ostreatus, exhibits anti-carcinoma activity,inhibits growth of bacteria and Fungi, and lowers cholesterol. Trametesversicolor produces heteroglucans with α-(1-4)- and β-(1-3) glycosidiclinkages with fucose in PSK (Krestin) and rhamnose and arabinose in PSP,have been shown to be anti-tumor and immunomodulatory. PSK, an approveddrug, is a protein which exhibits immunomodulating, anti-viral, andcholesterol regulating properties. Mycelial polysachharide extracts ofTremella fuciformis have been shown to be therapeutic for variouscirculatory disorders, to be neurologically healthy, anti-carcinoma,anti-tumor, and anti-aging.

Therefore, myceliation with Phellenis linteus, Polyporus umbellatus,Inonotus obliquus, Pleurotus ostreatus, Trametes versicolo, and/orTremella fuciformis (and any other fungal species described herein)andmyceliated cacao beans or other agricultural substrate productscontaining flavor and/or health promoting components derived fromPhellenis linteus, Polyporus umbellatus, Inonotus obliquus, Pleurotusostreatus, Trametes versicolor, and/or Tremella fuciformis (or any otherfungal species described herein) are also included in the instantinvention.

The amounts of flavor components or health promoting components added bythe fungal component as described herein can be estimated by one ofknowledge in the art, and includes up to 1 ng of the component per unitmyceliated cacao beans or other agricultural substrate product, or up to5 ng, up to 10 ng, up to 50 ng, up to 100 ng, up to 500 ng, up to 1 μg,up to 5 μg, up to 10 μg, up to 50 μg, up to 100 μg, up to 500 ug, up to1 mg, up to 2 mg, up to 5 mg, up to 10 mg, up to 20 mg, up to 50 mg, upto 100 mg, or up to 500 mg per unit myceliated cacao beans or otheragricultural substrate product. A unit of myceliated cacao beans orother agricultural substrate product can be variously defined as 1 g, 1lb, 1 kg, and the like.

Further Processing of Myceliated Product

In some embodiments, once fully myceliated, the myceliated cacao beansor other agricultural substrate product is optionally rinsed aftermyceliation. Rinsing may be performed to remove some or all parts of themycelia and/or other non-cacao beans or other non-agricultural substratematter.

In some embodiments, one fully myceliated, the myceliated product isoptionally dried. Drying can be accomplished by means known in the artfor drying cacao beans or other agricultural substrate. For example,myceliated product may be spread on a dry surface to dry. In oneembodiment, the myceliated product is dried down to about an 8 to 9%moisture content.

Optionally, the dried or undried myceliated product can be roastedand/or toasted by conventional methods known in the art. The optionalroasting step provides for deactivating the fungus, which may bedesirable to reduce the risk of mycoses.

Myceliated cacao can be subjected to known processing stepsnon-myceliated cacao. Accordingly the present invention can be used tomake myceliated cacao mass, myceliated cacao butter, myceliatedchocolate liquor, and all forms of chocolate derived thereof, as done bytempering, and other methods known in the art. The added benefits ofsuch food processing involve the unique nutritional profile (primarilythe polysaccharide and metabolite profile) imbued into these foods bythe myceliation.

In one embodiment of the invention, myceliated cacao bean extract isused for nutraceuticals in an extract form, or in a powdered formachieved from dehydrating the extract. In another embodiment themyceliated cacao bean extract is used as an additive in functional foodsto bolster flavor and nutrient content. This extract can be in aqueousform, infused in an alcohol base, or in vegetable glycerin, for example.A powdered form can be achieved from dehydrating the extract. Thepowdered form can be added to any packaged food. An example of afunctional food includes a bottled drink, a snack bar, or a high-proteinpowder mixture used to make protein shakes. The powdered form can alsobe used in ice-cream.

The myceliated product may optionally be extracted to prepare an extractfor use in food and/or drink products. For example, 1000 g of myceliatedroasted cacao bean or agricultural substrate may be fully extracted,with agitation; using 10 to 1000 ml of 121 to 122° C. pressurized wateras a buffer, containing 0.01% to 10% citric acid and 0.01 to 10%ascorbic acid. The resulting aqueous extract may be further purified andconcentrated using conventional methods. Myceliated cacao bean oragricultural substrate product extracts may be given an extended shelflife by methods known in the art such as formulation in 18% to 24%alcohol, 45% to 60% glycerol, or addition of 2.5 X volumes of honey orsimilar sugar such as maple syrup or evaporated cane sugar.

Myceliated cacao tastes sweeter than natural cacao because myceliumremoves bitter components from the bean. Accordingly, one benefit ofusing the myceliated cacao in functional foods is that a low sugar (orlow sweetener) version of any functional food including cacao can bemade to taste as good as compared with natural cacao having moresweeteners (i.e. sugar, stevia, or synthesized sweeteners). Also,myceliated cacao was found to be more alkaline than conventional cacao(unmyceliated), which provides advantages for digestion and health forthe consumer.

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention.

EXAMPLES

Example 1

Specific and pure strains of fungi obtained from referenced collectionswere manipulated in sterile environments in 1 to 10 gal plastic bags, 1qt to 1 gal glass jar, or on 10 cm to 15 cm petri plates, usingundefined, organic fruit and vegetable-based media including green cacaobeans or other agricultural substrate extract with 1.5% agar (w/v), inorder to monitor and ensure the general vigor and health of strains.

Mycelial samples were grown in a gentle, ambient sterile airflow for 2to 4 weeks, then excised from Petri plates and subsequently used forinoculation into liquid-state fermentation employing a similar undefinedfruit and vegetable-based media (but with no agar), using ambient air,in 1 qt to 1 gal glass jars. Some samples were grown in agitated andsome were grown in unagitated cultures in ambient air in stainless steeltanks designed for commercial beer brewing and/or fermentation, orcustom designed.

The unagitated liquid state fermentation formed a floating mass ofhyphae which exhibited continuous growth at interface of liquid and air.The mycelium of agitated and/or swirling cultures grew very quickly ashyphal spheres, which being hydrated, remained submerged, and had theappearance of gelatinous beads in small diameter. Hydrated hyphalspheres collapsed upon desiccation, wherein they were used forinoculating Petri-plates for strain propagation and quality control.

Sphere diameter in liquid-state fermentation was found to be inverselyproportional to agitation intensity and volume. Hyphal shear became moreefficient at higher agitation and swirling intensity, and once sheared,hyphae formed new spheres of smallest possible diameter, growing in sizeuntil they sheared again. When employed in continuous liquid-statefermentation, there existed a constant ratio of sphere diameters, andtherefore a constant supply of spheres on the order of microns wasproduced.

Thus, this example demonstrated that mycelia sphere diameter wasmanipulated for more efficient inoculation with inoculation efficiencybeing inversely proportional to sphere diameter.

Example 2

Mycelial cultures from unagitated liquid state fermentation (growthperiod of 2 to 4 weeks) formed a floating mass of hyphae, which weregently blended with a sharp, sterile cutting device prior to being usedfor inoculation. Gentle blending was achieved by mixing or lowhomogenization in a commercial blender in short bursts at slow speeds.Aliquots of blended liquid-state culture were used to inoculatesterilized unprocessed fruits and or vegetables, cereal grains, and/orculinary seed, or pasteurized culinary spice, medicinal herbs, naturalflavorings, tea mixes, green vanilla beans, green cacao beans, and cacaobeans or other agricultural substrate.

Example 3

Substrates for myceliation (containing both substrate and inoculatedmycelial culture) in jars or bags were gently mixed every few days untilthey commanded the substrate and became somewhat resistant to mixing orshaking, usually 2 to 4 weeks depending upon strain. The products werethen in a tempeh form. The myceliated green vanilla beans were cooked orbaked; the myceliated green cacao beans were baked or toasted; and themyceliated cacao beans or other agricultural substrate were toasted orroasted. Myceliated grain presented in tempeh form, or as an ingredientin food(s) including soups, stir fries, breads, and meat-substitutes,was made safe to eat, and bio-available, by cooking on low to mediumheat, 145° F. to 165° F., for 10 min to 60 min, at some point prior toconsumption. Other cultures in jars or bags, such as herbs and spiceswere dried at 100° F. to 145° F. for 1 h to 24 h, packaged and usedconventionally.

Myceliated honey formulations were stirred for 10 min to 90 min at 100°F. to 125° F., then poured into small glass bottles. Moreover,myceliated agricultural products were reformulated into value addedproducts such as egg noodles, meat substitutes, specialty flavorings,cooking sauces, soup ingredients and the like.

Example 4

For a large batch liquid-state and solid-state operation, pure cultureswere grown aerobically and inoculated into large industrial liquid-stateand large solid-state commercial processors operated continuously andaerobically for large-scale fermentation of food products. Aftercultures of media turned completely white or a representative colorthereof for a particular species, and had completely overgrown andcommanded the medium and were resistant to gentle mixing, the contentswere harvested, removed to plastic bags and refrigerated for quick useat either 40° F., or frozen for long-term storage, and subsequentutilization, at −20° F. Fermented media were prepared into gourmet humanfoods including: “tempeh style” meat substitutes, egg-noodles, specialtyflavorings, breads, extracts and cooking-sauces, or used directly as afresh ingredient in soup and/or stir fried recipes, or packaged.

Example 5

Agricultural substrates were completely myceliated by inoculating withpure cultures of fungal strains selected from A. blazei, C. sinensis, G.lucidum, H. erinaceus, G. frondosa, P. eryngii, P. ostreatus, P.citrinopileatus, P. djamor, T versicolor, L. edodes, F. velutipes, V.volvacea, H. marmoreus, P. nameko, I. obliquus, M. hortensis, P.umbellatus, and T. fuciformis were subjected to heat treatment 1 hour to24 hours prior to harvest for 1 min to 2 hours at 145° F. to 195° F.followed by recovery at room temperature for 45 min to 48 hours.

Example 6

71 lbs. of cacao beans were soaked in an equivalent volume of water for5 minutes, after which the water was filtered off and the cacao wasequally distributed into 10 polypropylene bags with 0.2 μm breatherpatches. The tops of the bags were folded over the bag and had rubberbands wrapped around each side of the bag. The bags were sterilized for140 minutes at 22 psi with slightly dry saturated steam at 255° F. Thebags were then cooled in a sterile environment for 8 hours, and weresubsequently inoculated with submerged liquid tissue cultures ofGanoderma lucidum, Cordyceps sinensis, I. obliquus, or Morchellaangusticeps, with sterile tools and in sterile operation inside asterile laminar flow hood. The inoculant were grown in 4 L flasks, in1.5 L of media prepared from the mixture with 10 L of organic potatoextract, 2 L of raw cacao bean extract, and 1 L organic mango juice, and50 mL of inoculant was used for each bag of cacao after the inoculanthad grown for 7 days under 60 RPM of 1 in. radius agitation. Thecultures myceliated for two weeks, whereupon they were dried and tasted.It was noted that the H. erinaceus and I. obliquus cacao conferred theleast bitter taste (most improved taste).

Example 7

Samples of Barley, Brown Rice, Buckwheat, Bulgur (cracked wheat),flaxseed, Grano, Millet, Oats, Oat Breat, Oat Cereal, Oatmeal, Popcorn,Whole Wheat Cereal Flakes, Muesli, Rolled Oats, Quinoa, Rye, Sorghum,Spelt, Triticale, Whole Grain Barley, Wheat Berries, Whole GrainCornmeal, Whole Rye, Whole Wheat Bread, Whole Wheat Couscous, and WildRice were individually mixed with half their volume of water inappropriately prepared quart ball jars or autoclavable bags withbreather patches and sterilized at 15 psi for 90 minutes or 22 psi for80 minutes. Once cool, the grain jars were inoculated with half to wholecolonies of floating or submerged fungal cultures selected from one ofthe following: Ganoderma lucidum, Cordyceps sinensis, Hericiumerinaceus, Agaricus blazei, Grifola frondosa, Pleurotus ostreatus,Trametes versicolor, Laetiporus sulphureus, Flammulina velutipes,Lentinula edodes, Morchella angusticeps, Morchella crassipes, Morchellahesculenta, Tremella fuciformis, and Inonotus obliquus, where thefloating cultures were grown on an organic undefined media in quart orhalf-gallon ball jars, and where the submerged cultures were grown in 4L flasks in 1.5 L of undefined media. After myceliating for two weeks,the jars were thoroughly grown through, and were pasteurized at 212° F.in a double boiler for 7 minutes before being cooked and consumed astempeh, or further formulated into a nutriceutical (i.e. formulated into2 gallons honey in association with the small molecules ascorbic acid (1g) and citric acid (10 g) with 8 mL of orange and tangerine essentialoils with). This formulation step began by blending the pasteurizedculture with 2 liters of water, or not, and then the ascorbic and citricacid was added to the blend, then the essential oils, then the honey,the formulation was then heated in a double boiler, bottled, andpasteurized once bottled to ensure a secure seal.

Example 8 Small Batch Work-Cacao

48 lbs. of cacao beans were divided into 48 equal portions in cleanquart ball jars with lids constructed to enable gaseous diffusion past acollar. These 48, 1 lbs. masses of cacao beans were soaked with ¾ quartof water for two hours. The water in the mixtures was filtered off. Thejars were then subjected to 90 minutes of sterilization temperatures at15 psi, and placed in a sterile laminar air flow to cool for 8 hours.Once cool, the cacao beans were inoculated with half to whole coloniesof fungus selected from one of the following: Ganoderma lucidum,Cordyceps sinensis, Hericium erinaceus, Agaricus blazei, Grifolafrondosa, Pleurotus ostreatus, Trametes versicolor, Laetiporussulphureus, Flammulina velutipes, Lentinula edodes, Morchellaangusticeps, Morchella crassipes, Morchella hesculenta, Tremellafuciformis, and Inonotus obliquus, doing three of each, growing on anundefined vegetable and fruit juice agar media containing green cacaoextract as described, with sterile tools and in sterile operation insidethe laminar flow hood. The cultures myceliated for 7 to 21 days, withsamples of each being pulled out for drying and roasting at the 7^(th),14^(th), and 21^(st) days. The smell of the culture and taste of thecacao at the 7^(th) day indicated that the cultures were complete,though longer myceliation periods yielded greater cell mass.

Large Batch Work-Cacao

528 lbs. of cacao beans or other agricultural substrate were soaked intwo different procedures. In the first procedure, the beans were soakedthree times, for 20 minutes each soak, in the second procedure, thebeans were soaked for 20 minutes through a constant stream of filteredwater. The beans were then packed into polypropylene bags with 0.2micron breather patches, with the tops of the bags folded over withrubber bands wrapped around the sides of the bags, such that steam andgas diffusion could occur through breather patch and through the foldedsides of the bags. The bags were sterilized under a liquid cycle at 22psi for 80 minutes, and then allowed to cool for 8 hours. The bags wereinoculated with fungi from the following species: Ganoderma lucidum,Cordyceps sinensis, I. obliquus, and Morchella angusticeps. TheGanoderma lucidum culture was grown in a bioreactor, with 10 L oforganic potato extract, 2 L of green coffee extract, and 1 L organicmango juice diluted to 100 liters with RO water. The bioreactor wassparged with compressed air filtered through two inline 0.2 micronhydrophobic capsule filters, and the reactor was kept under 2-3 psithrough the use of check valves on the air supply and venting lines with2-3 psi cracking pressure ratings. The inoculant was readily grown in 48hours, and was harvested through a diaphragm valve located at the bottomof the reactor, which led to a harvesting line that had teed and valvedoff access to a steam line and steam trap, with an inline check valve,through six feet of flexible stainless steel hosing, to a solenoid valveconnected to a timer and foot switch, followed by a flow metering valveto an elbowed sanitary fitting. While being steamed, the elbowedsanitary fitting was connected to a ball valve that connected to thesteam exhaust manifold. The ball valve was closed after steaming theline, and the ball valve was detached from the harvesting line onceentered into a laminar flow hood, so as to keep the whole line sterile.The Cordyceps sinensis, Tuber melanosporum, and Morchella angusticepscultures were grown in 4 L flasks, in 1.5 L of the same media used inthe bioreactor pre-dilution. These cultures were grown for six days, andwere used to inoculate the bags of sterilized cacao beans or otheragricultural substrate. The beans were myceliated for 7 days, wheretheir smell conferred the desired taste profile of the beverage madefrom the roasted myceliated beans, whereupon they were dried on the8^(th) day to a 13% moisture content.

Example 9

A suitable fungi for use in the methods of the present invention wasprepared by the following methods. The following G. lucidum strains werepurchased commercially from the Pennsylvania State University mushroomculture collection: 496 Ling ZHI; Singapore commercial line; July, 1985;⊚IFO #8436; IFO-Japan; July 30, 1985; 510 Red oak, State College, Pa.;D. J. Royse; September, 1985; 549 Y. H. Park, ASI-Korea; Dec. 5, 1985;550 Y. H. Park, ASI-Korea; Dec. 5, 1985; 551 Y. H. Park, ASI-Korea; Dec.12, 1985; 580 Y. H. Park, ASI-Korea; Feb. 2, 1985; 607 Y. H. Park,ASI-Korea; Feb. 19, 1985; 617 Y. H. Park, ASI-Korea; Feb. 25, 1985; 618Y. H. Park, ASI-Korea; Feb. 25, 1985; 619 Y. H. Park, ASI-Korea; Feb.25, 1985; 620 Y. H. Park, ASI-Korea; Feb. 25, 1985; 621 Y. H. Park,ASI-Korea; Feb. 25, 1985; 622 Y. H. Park, ASI-Korea; Feb. 25, 1985; 623Y. H. Park, ASI-Korea; Feb. 25, 1985; 624 Y. H. Park, ASI-Korea; Feb.25, 1985; 625 Y. H. Park, ASI-Korea; Feb. 25, 1985; 626 Y. H. Park,ASI-Korea; Feb. 25, 1985; 627 Y. H. Park, ASI-Korea; Feb. 25, 1985; 665Quimio; Philippines; Mar. 6, 1986; 669 Y. H. Park, ASI-Korea; Mar. 25,1986; 686 B. W. Yoo; Apr. 28, 1986; 724 T. Mitchel, Lawn PSU ForestryBldg. Sep. 16, 1990; 806 Alice Chen; Buffalo, N.Y.; April, 1994; 807Alice Chen; N. C.; April, 1994; 841 White Oak; PSU Campus; J. Peplinski;August, 1999. The above strains were cultured using the media describedherein comprising cacao beans or other agricultural substrates extract(see Example (10). Many strains were unable to grow and/or died on themedia. Surprisingly, the inventors found that G. lucidum strain 806Alice Chen; Buffalo, N.Y. was able to grow on the media comprising greencoffee bean extract and was selected for further use in accordance withthe instant invention.

Example 10

Fungi (including G. lucidum strain 806, C. sinensis strain859, asdescribed herein, also H. erinaceus, T versicolor, L. edodes, Tmatsutake, F. velutipes, A. blazei, G. frondosa, P. nameko, L.officinalis, M. hortensis, M. angusticeps, A. auricula, T fuciformis, I.obliquus, F. fomentarius, L. sulfureus) were maintained on a culturecomprising an undefined media including extract of cacao beans.Experiments showed that use of the media including extract of cacaobeans to culture the maintained the fungi's ability to tolerate, growon, metabolize, remove or reduce caffeine or undesirable flavorcomponents. It was also found that successive propagations of fungi asdefined above caused enhancement and/or improvement of the fungi'sability to tolerate, grow on, metabolize, remove or reduce caffeine ordecrease undesirable flavor components, resulting in training oradapting the fungi to undefined media including extract of cacao beans.Such fungi with changed, improved, and adapted properties as describedherein, relative to the starting strains, either selected or unselected,were developed. These adapted strains were deposited with the ATCC asdescribed elsewhere herein.

The undefined media including extract of cacao beans was made asfollows: 2 lbs cacao beans or other agricultural substrate, pulverizedwas mixed with ¼ gallon water at room temperature. The mixture wasallowed to extract for 20 minutes with shaking, then filtered threetimes through fine mesh. Separately, 5 organic potatoes were placed in10 L of water and autoclaved 20 minutes to soften the potatoes. Thepotatoes were then pulverized with a potato masher, and then filteredthrough fine mesh three times. 1 L of commercial unsweetened fruit juicewas be added. These solutions are combined and autoclaved. This recipewas also scaled up or down as required.

The washed cacao beans were washed in water and the moisture content wasraised to about 30%. At other times the moisture content was raised toabout 60%.

Liquid culture: The culture comprising fungi for use in inoculating theprepared cacao beans or other agricultural substrate was agitated withsparged air and a motorized paddle to create turbulent environment andto shear hyphae with pure mechanical force. The dual agitation methodwas superior to either method individually, since sparged air createdthe most turbulence at the top half of the culture, while affecting thebottom less, which was agitated by a motorized paddle. In return thepaddle could be run at a lower RPM and still obtain the hyphal spheresize obtained by a faster RPM in the absence of sparging. The hyphalsize was about 2-5 micron in diameter). Undamaged mycelium and propermorphology in the prepared fungi were prepared by this method and usedfor culturing and/or myceliation.

Example 11

80 kg of provided green cacao beans from the Ivory Coast were dispensedinto a food-grade 5 gallon bucket 5 kg at a time, and RO water was addedto fully submerge the beans. After soaking for 5 minutes, the contentsof the bucket were poured through a fine stainless-steel mesh colanderand the wet beans were recovered. After each soak, the beans were placedinto clean 2.2 mm polypropylene bags with dimensions of 5″×8″×19″(depth×width×height), outfitted with a 2″×3″ 0.2 μm filter patch, 2.2 kgof beans per bag. The bags were gently rolled up so as not to invert thecontents of the bag, and loosely wrapped with EPDM bands. The 36prepared bags were loaded into an autoclave and sterilized on a 140minute liquid cycle with 122-124° C. slightly superheated steam. Thebags were then taken out of the autoclave and stored in a clean-space tocool for 12 hours. At this point, the beans were inoculated with 200 mLof submerged liquid tissue cultures of Ganoderma lucidum, Cantharelluscibarius, Morchella angusticeps, Tricholoma matsutake, or Grifolafrondosa, and sealed with a bag-sealer. These cultures had been preparedas 1.5 L cultures in 4 L Erlenmeyer flask. The culture media comprised 4g/L organic potato starch powder, 0.4 g/L organic carrot powder, and 10%(v/v) aqueous cacao bean extract. The aqueous cacao bean extract hadbeen prepared by soaking 1 lb of cacao beans in 2 gallons of water forhalf an hour at room temperature, and collecting the filtrate through 3filtrations through a fine mesh colander. 3 flasks were prepared foreach strain, and they were sterilized for 130 minutes on a liquid cyclewith 122-124° C. slightly superheated steam. These flasks wereinoculated from Petri plates that had been grown on media comprising 8g/L organic potato starch, 0.6 g/L organic carrot powder, 33% (v/v)organic mango puree, and 18 g/L agar. The submerged liquid cultures grewfor 7 days and shook at 120 RPM on a 1″ diameter swing radius. Theinoculated cacao bean cultures myceliated for 21 days, at which pointthe cultures were dried to an 8 to 9% moisture content. The beans wereuniformly myceliated, with a sheath of white around every shell. Thebeans were then sent off to a professional chocolate manufacturingcompany for formulation into chocolate.

Example 12

20 kg of provided green CCN51 cacao beans were split into 2, 9 kgsamples, with 2 kg being kept back as a control. Each 9 kg sample wasdispensed into a food-grade 5 gallon bucket and soaked in RO water for10 minutes. The beans were subsequently recovered through filtrationthrough a fine stainless-steel mesh colander, and dispensed into clean 2L Wheaton flasks, 1 kg at a time. The flasks were outfitted withtin-coil collars as is known in the art. These 18 flasks were sterilizedfor 120 minutes on a gravity cycle with 121-122° C. saturated steam, andcooled in a clean space for 8 hours.

Before inoculation, 50 mL of 2-methoxy-3-isopropylpyrazine was added to2 cultures, 100 mL of 2-methoxy-3-isopropylpyrazine added to another 2cultures, 150 mL of 2-methoxy-3-isopropylpyrazine added to another 2cultures, 200 mL of 2-methoxy-3-isopropylpyrazine added to another twocultures, and 250 mL of 2-methoxy-3-isopropylpyrazine added to another 2cultures. These 8 cultures were then inoculated, 4 with submerged liquidtissue cultures of Ganoderma lucidum and 4 with submerged liquid tissuecultures of Cordyceps sinensis. The other 10 cultures were inoculated ina similar manner, 5 of G. lucidum and 5 of C. sinensis. These cultureswere made from media comprising 8 g/L organic potato starch powder and0.6 g/L organic carrot powder, and were 1.5 L in volume in 4 LErlenmeyer flasks. These cultures had been inoculated from Petri platescomprising 40% (v/v) organic mango puree, and grew for 4 days on ashaker table at 120 RPM with a 1″ diameter swing radius. The cultureswith no additional 2-methoxy-3-isopropylpyrazine myceliated for 21 days,at which point they were dried for 2 days to an 8 to 9% moisturecontent. The cultures comprising 2-methoxy-3-isopropylpyrazine tooklonger to myceliate, and went for 40 days. Before drying, beans fromeach sample were plated out onto 10 plates with media comprising 20%(v/v) organic potato extract and 15% (v/v) organic carrot extract, so asto isolate the 2-methoxy-3-isopropylpyrazine adapted strains. 80 platesin total were made. The remaining beans were dried over 2 days to an 8to 9% moisture content, though were not formulated, as the lingering2-methoxy-3-isopropylpyrazine smell was determined too intense for food.The isolated strain was subsequently used to myceliate more providedCCN51 cacao beans in an identical manner as discussed above, except thatthe myceliation went for 21 days, as for the non-adapted strain. 5people tasted the formulated chocolate, and there was a markeddifference between the flavor of the 2-methoxy-3-isopropylpyrazineadapted strain as compared to the non-adapted. Both strains of eachspecies generated an edible CCN51 bean (the control was determined to becompletely non-palatable), though the 2-methoxy-3-isopropylpyrazineadapted strain created a richer, almost buttery type flavor.

Example 13

15 kg of provided Ecuadoran green cacao beans were dispensed into 14 2.2mm polypropylene bags at 1 kg/bag. 1 kg of provided green cacao beanswere held back as a control. The bags had dimensions of 4″×5″×19″(depth×width×height), and were outfitted with a 3″×2″ 0.2 μm breatherpatch. 500 mL of RO water was added to each bag. The bags were carefullyrolled-up so as not to invert the contents of the bag, and looselywrapped with EPDM bands. The bags were placed into an autoclave andsterilized for 120 minutes on a liquid cycle with 121-122° C. saturatedsteam. Once the cycle finished, the bags were moved into a clean-spaceand cooled for 15 hours, at which time they were inoculated, half with400 mL of submerged liquid tissue cultures of Hericium erinaceus andhalf with 400 mL of submerged liquid tissue culture of Inonotusobliquus. The cultures had media comprised of 4 g/L organic potatostarch, 0.4 g/L organic carrot powder, and had 15 g of cacao beans addedto the 1.5 L volume before being sterilized. The 4 L Erlenmeyer flaskscontaining the sterilized media were inoculated from Petri plates. TheH. erinaceus cultures myceliated for 21 days, but the I. obliquuscultures were fully colonized by day 7. The samples were then dried for3 days to a 7-8% moisture content, and 2.5 kg of each sample wasformulated into chocolate over the course of 5 days. Another 2 days werespent formulating the control sample into chocolate. The formulatedmyceliated chocolate was tasted by 5 people, and the results confirmedthat these two fungal species effect wonderful flavor changes on cacao.Both had distinct buttery flavors, the H. erincaceus sample had a uniquealmond smell, while the I. obliquus sample was the smoother of the two.The control sample was determined to be too bitter to eat.

Example 14

0.5 kg of provided Nigerian green cacao beans were soaked in a 2 gallonstainless-steel kettle for half an hour, at which point the beans werecollected through a fine stainless-steel mesh filtration. The hydratedcacao beans were placed into 2 separate 1 L Wheaton jars, which weresubsequently outfitted with tin-foil collars. The jars were placed intoan autoclave and sterilized for 120 minutes, upon which time they wereplaced into a clean-space to cool. Three days later they were inoculatedwith Ganoderma lucidum from Petri plates. A 1 in² piece of a Petri platewas used for one sample, while the rest of the plate was used for theother. The Petri plates were grown on a medium comprising aqueousorganic potato extract and organic mango puree. The samples eachmyceliated for 60 days. An interesting growth cycle occurred, where thefungi in the sample from the 1 in² piece of Petri plate inoculant wentthrough three separate log phases to fully colonize the culture. Theother culture myceliated fully in 22 days. Both samples went for 60days, at which point in the time the mycelium was thick and fuzzy, andmost likely past the point of palatable consumption, the inventors beingconvinced from the smell that the chocolate made from such samples wouldhave an overwhelming fungal taste.

Example 15

The coffee beans and grounds produced by the methods of the instantinvention contained added polysaccharides and p glucans. An analysisshowed that Robusta coffee grounds produced by the methods of theinvention had 30.54 mg dextran per gram of coffee grounds. This resultprovided the total polysaccharide amount in the substrate through aspectrophotometric method based on a modified phenol-sulfuric acidapproach. The analysis also showed that Robusta coffee grounds producedby the methods of the invention had β glucans at 0.432%, as measured bythe MYBG method utilizing strong hydrolysis conditions to hydrolyze βglucan with quantification by spectrophotometric method. This representsan advantage over consuming beta-glucans from Reishi mushrooms, as thesemushrooms are a non-culinary mushroom for reasons of bitterness,woodiness, and hardness, or in pill form.

Example 16 Taste Comparison 1, G. Lucidum on Ivory Coast Cacao Beans

A chocolate company out of southern France, confirmed the flavorenhancing effects of myceliation by G. lucidum on their own cacao beanssent to the inventors to experiment with. Their trained tasters sampledmyceliated and normal (unmyceliated) cacao products in blind tastetests. Notes were taken and detailed analyses were conducted. Bitternesswas reduced by approximately 50% and desirable nutty flavors wereincreased.

Taste Comparison 2 Cacao Beans

Cacao beans prepared by the method of the invention were processed intochocolate and tasted to compare to chocolate made by beans that had notbeen subjected to instant processes. The taster stated that thechocolate from the myceliated chocolate had an aroma and taste that wasricher, smoother, and sweeter with less bitter, harsh, and/or acidictastes and aromas than compared with the chocolate made from theuntreated cacao beans. The taster stated that less sugar and/or othersweetener such as dairy were necessary to create a chocolate that washighly palatable as compared to chocolate made from the untreated cacaobeans.

What is claimed is:
 1. A method for preparation of a myceliated cacaoproduct, comprising: a) providing prepared cacao beans comprising thesteps of: i. providing cacao beans; ii. sterilizing the prepared cacaobeans; b) providing a prepared fungal component; c) inoculating theprepared cacao beans with the prepared fungal component; and d)culturing the prepared cacao beans and prepared fungal component toallow myceliation to prepare the myceliated cacao product.
 2. The methodof claim 1, wherein the method reduces the amount of at least oneundesirable taste component.
 3. The method of claim 2, wherein theundesirable taste component is 2-methoxy-3-isopropylpyrazine.
 4. Themethod of claim 2, wherein the undesirable taste component is amethylxanthine, catechin, epicatechin, or gallic acid equivalent.
 5. Themethod of claim 1, wherein the step of providing prepared cacao beansfurther comprises hydrating the cacao beans.
 6. The method of claim 5,wherein the cacao beans are hydrated to about a 60% moisture level or toabout a 30% moisture level.
 7. The method of claim 1, wherein theprepared fungal component is selected from the group consisting ofHericium erinaceus, Pleurotus ostreatus, Pleurotus eryngii, Pleurotuscitrinopileatus, Pleurotus djamor, Trametes versicolor, Lentinulaedodes, Armillariella mellea, Tricholoma matsutake, Flammulinavelutipes, Volvariella volvacea, Agaricus campestris, Agaricus blazei,Grifola frondosa, Pholiota nameko, Agrocybe cylindracea, Boletus edulis,Ganoderma lucidum, Ganoderma applanatum, Hypsizygus marmoreus, Morchellahortensis, Morchella angusticeps, Morchella esculenta, Phellinuslinteus, Auricularia auricula, Tremella fuciformis, Inonotus obliquus,Fomes fomentarius, Laetiporus sulfureus, Cordyceps sinensis, Cordycepsmilitaris, Cantharellus cibarius, Polyporus umbellatus, and combinationsthereof.
 8. The method of claim 7, wherein the prepared fungal componentis G. lucidum.
 9. The method of claim 8, wherein the prepared fungalcomponent is H. erinaceus.
 10. The method of claim 7, wherein theprepared fungal component is C. sinensis.
 11. The method of claim 7,wherein the prepared fungal component is I. obliquus.
 12. The method ofclaim 1, wherein the prepared fungal component is prepared by a methodcomprising screening a number of strains of fungi and selecting a strainhaving an enhanced ability to tolerate, grow on, metabolize or utilizecacao beans.
 13. The method of claim 1, wherein the prepared fungalcomponent is prepared by a method comprising maintaining a strain offungi on an undefined media comprising an aqueous cacao bean extract andan energy source.
 14. The method of claim 1, wherein the prepared fungalcomponent is prepared by a method comprising maintaining a strain offungi on an undefined media comprising a methylxanthine, catechin,epicatechin, or gallic acid equivalent or 2-methoxy-3-isopropylpyrazine,and an energy source.
 15. The method of claim 14, wherein themaintenance of the strain of fungi causes adaptation of the fungiresulting in enhancement of the fungi's ability to tolerate, grow on,metabolize or utilize cacao beans or other agricultural substrate. 16.The method of claim 1, wherein the culturing step is carried out forabout 7 days.
 17. The method of claim 1, wherein the myceliated producthas a reduced level of at least one undesirable taste component and anincreased level of at least one myceliation product, relative to thecacao beans.
 18. The method of claim 17, wherein the myceliation productis a β-glucan, pyrazine, or polysaccharides.
 19. A myceliated productprepared by the method of claim 1.